Home > To define functional network for calcineurin the conserved Ca2+ calmodulin regulated phosphatase we systematically identified its substrates in cerevi

To define functional network for calcineurin the conserved Ca2+ calmodulin regulated phosphatase we systematically identified its substrates in cerevi

To define functional network for calcineurin the conserved Ca2+ calmodulin regulated phosphatase we systematically identified its substrates in cerevisiae using phosphoproteomics and bioinformatics followed by copurification and dephosphorylation assay this study establishes new calcineurin functions and reveals mechanisms that shape calcineurin network evolution analyses of closely related yeasts show that many protein were recently recruited to the network by acquiring calcineurin recognition motif calcineurin substrates in yeast and mammals are distinct due to network rewiring but surprisingly are phosphorylated by similar kinases We postulate that corecognition of conserved substrate features including phosphorylation and docking motifs preserves calcineurin kinase opposition during evolution one example we document is composite docking site that confers substrate recognition by both calcineurin and MAPK We propose that conserved kinase phosphatase pairs define the architecture of signaling networks and allow other connections between kinases and phosphatases to develop that establish common regulatory motifs in signaling networks

purpose beagle dogs are used to study oral pharmacokinetics and guide development of drug formulations for human use since mechanistic insight into species differences is needed to translate findings in this species to human abundances of cytochrome P450 CYP and uridine diphosphate glucuronosyltransferase UGT drug metabolizing enzymes have been quantified in dog liver and intestine method abundances of enzymes were measured in beagle dog intestine and liver using selected reaction monitoring mass spectrometry results seven and two CYPs were present in the liver and intestine respectively CYP3A12 was the most abundant CYP in both tissues seven UGT enzymes were quantified in the liver and seven in the intestine although UGT1A11 and UGT1A9 were present only in the intestine and UGT1A7 and UGT2B31 were found only in the liver UGT1A11 and UGT1A2 were the most abundant UGTs in the intestine and UGT2B31 was the most abundant UGT in the liver summed abundance of UGT enzymes was similar to the sum of CYP enzymes in the liver whereas intestinal UGTs were up to four times more abundant than CYPs the estimated coefficients of variation of abundance estimates in the livers of donors were separated into biological and technical components which ranged from to and to respectively conclusions abundances of canine CYP enzymes in liver and intestine have been confirmed in larger number of dogs and UGT abundances have been quantified for the first time the biological variability in hepatic CYPs and UGTs has also been estimated

the study of protein and protein complexes using chemical crosslinking followed by the MS identification of the cross linked peptide has found increasingly widespread use in recent years thus far such analyses have used almost exclusively homobifunctional amine reactive cross linking reagents here we report the development and application of an orthogonal cross linking chemistry specific for carboxyl groups chemical cross linking of acidic residue is achieved using homobifunctional dihydrazides as cross linking reagents and coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes In addition to cross links formed through insertion of the dihydrazides with different spacer lengths zero length cross link products are also obtained thereby providing additional structural information We demonstrate the application of the reaction and the MS identification of the resulting cross linked peptide for the chaperonin TRiC CCT and the 26S proteasome the results indicate that the targeting of acidic residue for cross linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross linking thereby expanding the yield of structural information that can be obtained from cross linking studies and used in hybrid modeling approaches

background tight spatio temporal signaling of cytoskeletal and adhesion dynamics is required for localized membrane protrusion that drives directed cell migration different ensembles of protein are therefore likely to get recruited and phosphorylated in membrane protrusions in response to specific cues results here we use an assay that allows to biochemically purify extending protrusions of cell migrating in response to three prototypical receptors integrins recepor tyrosine kinases and coupled protein receptors using quantitative proteomics and phospho proteomics approaches we provide evidence for the existence of cue specific spatially distinct protein networks in the different cell migration modes conclusions the integrated analysis of the large scale experimental data with protein information from databases allows us to understand some emergent properties of spatial regulation of signaling during cell migration this provides the cell migration community with large scale view of the distribution of protein and phospho protein regulating directed cell migration

bulk degradation of cytoplasmic material is mediated by highly conserved intracellular trafficking pathway termed autophagy this pathway is characterized by the formation of double membrane vesicles termed autophagosomes engulfing the substrate and transporting it to the vacuole lysosome for breakdown and recycling the atg1 ULK1 kinase is essential for this process however little is known about its targets and the means by which it controls autophagy here we have screened for atg1 kinase substrates using consensus peptide arrays and identified three components of the autophagy machinery the multimembrane spanning protein atg9 is direct target of this kinase essential for autophagy phosphorylated atg9 is then required for the efficient recruitment of atg8 and atg18 to the site of autophagosome formation and subsequent expansion of the isolation membrane prerequisite for functioning autophagy pathway these findings show that the atg1 kinase acts early in autophagy by regulating the outgrowth of autophagosomal membranes

A major goal in proteomics is the comprehensive and accurate description of proteome this task includes not only the identification of protein in sample but also the accurate quantification of their abundance although mass spectrometry typically provides information on peptide identity and abundance in sample it does not directly measure the concentration of the corresponding protein specifically most mass spectrometry based approaches shotgun proteomics or selected reaction monitoring allow one to quantify peptide using chromatographic peak intensities or spectral counting information ultimately based on these measurements one wants to infer the concentrations of the corresponding protein inferring properties of the protein based on experimental peptide evidence is often complex problem because of the ambiguity of peptide assignments and different chemical properties of the peptide that affect the observed concentrations We present SCAMPI novel generic and statistically sound framework for computing protein abundance scores based on quantified peptide In contrast to most previous approaches our model explicitly includes information from shared peptide to improve protein quantitation especially in eukaryotes with many homologous sequence the model accounts for uncertainty in the input data leading to statistical prediction intervals for the protein scores furthermore peptide with extreme abundances can be reassessed and classified as either regular data points or actual outliers We used the proposed model with several datasets and compared its performance to that of other previously used approaches for protein quantification in bottom up mass spectrometry

candida albicans public proteomic datasets though growing steadily in the last few years still have very limited presence in online repositories We report here the creation of albicans peptideatlas comprising near distinct peptide at false discovery rate FDR that account for over canonical protein at FDR based on data from experiments we attained coverage of of the albicans open reading frame sequence ORFs in the database used for the searches this peptideatlas provides several useful features including comprehensive protein and peptide centered search capabilities and visualization tools that establish solid basis for the study of basic biological mechanisms key to virulence and pathogenesis such as dimorphism adherence and apoptosis further it is valuable resource for the selection of candidate proteotypic peptide for targeted proteomic experiments via selected reaction monitoring SRM or SWATH MS biological significance this albicans peptideatlas resolves the previous absence of fungal pathogens in the peptideatlas project It represents the most extensive characterization of the proteome of this fungus that exists up to the current date including evidence for uncharacterized ORFs through its web interface peptideatlas supports the study of interesting protein related to basic biological mechanisms key to virulence such as apoptosis dimorphism and adherence It also provides valuable resource to select candidate proteotypic peptide for future SRM targeted proteomic experiments this article is part of special issue entitled trends in microbial proteomics

mitochondrial ribosomes synthesize number of highly hydrophobic protein encoded on the genome of mitochondria the organelles in eukaryotic cell that are responsible for energy conversion by oxidative phosphorylation the ribosomes in mammalian mitochondria have undergone massive structural changes throughout their evolution including ribosomal RNA shortening and acquisition of mitochondria specific ribosomal protein here we present the three dimensional structure of the 39S large subunit of the porcine mitochondrial ribosome determined by cryo electron microscopy at resolution the structure combined with data from chemical crosslinking and mass spectrometry experiments reveals the unique features of the 39S subunit at near atomic resolution and provides detailed insight into the architecture of the polypeptide exit site this region of the mitochondrial ribosome has been considerably remodelled compared to its bacterial counterpart providing specialized platform for the synthesis and membrane insertion of the highly hydrophobic protein components of the respiratory chain

relating protein concentration to cell type specific responses is one of the remaining challenges for obtaining quantitative system level understanding of mammalian signaling here we used mass spectrometry MS and antibody based quantitative proteomic approaches to measure protein abundances for of hand curated reconstructed erbB network of protein in two established cell types HEK293 and MCF and in primary keratinocyte cell comparison with other quantitative studies allowed building set of erbB network protein expressed in all cell and another which are cell specific and could impart specific properties to the network As proof of concept of the importance of protein concentration we generated small simplified mathematical model encompassing ligand binding followed by receptor dimerization activation and degradation the model predicts erbB phosphorylation in HEK293 MCF and keratinocyte cell simply by incorporating cell type specific erbB1 erbB2 and caveolin abundances but otherwise contains similar rate constants altogether the data provide resource for protein abundances and localization to be included in larger mathematical models enabling the generation of cell type specific computational models MS data have been deposited to the proteomexchange via PRIDE with identifier PXD000623 and PASSEL with identifier PASS00372

the PRIME XS consortium is pan european infrastructure for proteomics As prologue to this special issue of molecular cellular proteomics on the research activities of the PRIME XS consortium we as the guest editors of this issue provide an overview of the structure and activities of this consortium which is funded by the european union 7th framework programme for research and technological development

pyopenMS is an open source python based interface to the C++ openMS library providing facile access to feature rich open source algorithm library for MS based proteomics analysis It contains python bindings that allow raw access to the data structures and algorithms implemented in openMS specifically those for file access mzXML mzML traML mzidentML among others basic signal processing smoothing filtering de isotoping and peak picking and complex data analysis including label free SILAC iTRAQ and SWATH analysis tools pyopenMS thus allows fast prototyping and efficient workflow development in fully interactive manner using the interactive python interpreter and is also ideally suited for researchers not proficient in C++ In addition our code to wrap complex C++ library is completely open source allowing other projects to create similar bindings with ease the pyopenMS framework is freely available at https pypi python org pypi pyopenms while the autowrap tool to create cython code automatically is available at https pypi python org pypi autowrap both released under the clause BSD licence

We describe method that integrates data derived from different mass spectrometry MS based techniques with modeling strategy for structural characterization of protein assemblies We encoded structural data derived from native MS bottom up proteomics ion mobility MS and chemical cross linking MS into modeling restraints to compute the most likely structure of protein assembly We used the method to generate near native models for three known structures and characterized an assembly intermediate of the proteasomal base

the identification of biomarkers indicating the level of aggressiveness of prostate cancer PCa will address the urgent clinical need to minimize the general overtreatment of patients with non aggressive PCa who account for the majority of PCa cases here we isolated formerly linked glycopeptides from normal prostate and from non aggressive aggressive and metastatic PCa tumor tissues and analyzed the samples using SWATH mass spectrometry an emerging data independent acquisition method that generates single file containing fragment ion spectra of all ionized species of sample the resulting datasets were searched using targeted data analysis strategy in which an priori spectral reference library representing known glycosites of the human proteome was used to identify groups of signals in the SWATH mass spectrometry data On average we identified glycosites from each sample out of those glycoproteins showed significant quantitative changes associated with diverse biological process involved in PCa aggressiveness and metastasis and indicated functional relationships two glycoproteins acylethanolamine acid amidase and protein tyrosine kinase that were significantly associated with aggressive PCa in the initial sample cohort were further validated in an independent set of patient tissues using tissue microarray analysis the results suggest that acylethanolamine acid amidase and protein tyrosine kinase may be used as potential tissue biomarkers to avoid overtreatment of non aggressive PCa

quantifying the similarity of spectra is an important task in various areas of spectroscopy for example to identify compound by comparing sample spectra to those of reference standards In mass spectrometry based discovery proteomics spectral comparisons are used to infer the amino-acid sequence of peptide In targeted proteomics by selected reaction monitoring SRM or SWATH MS pre determined sets of fragment ion signals integrated over chromatographic time are used to identify target peptide in complex samples In both cases confidence in peptide identification is directly related to the quality of spectral matches In this study we used sets of simulated spectra of well controlled dissimilarity to benchmark different spectral comparison measures and to develop robust scoring scheme that quantifies the similarity of fragment ion spectra We applied the normalized spectral contrast angle score to quantify the similarity of spectra to objectively assess fragment ion variability of tandem mass spectrometric datasets to evaluate portability of peptide fragment ion spectra for targeted mass spectrometry across different types of mass spectrometers and to discriminate target assay from decoys in targeted proteomics altogether this study validates the use of the normalized spectral contrast angle as sensitive spectral similarity measure for targeted proteomics and more generally provides methodology to assess the performance of spectral comparisons and to support the rational selection of the most appropriate similarity measure the algorithms used in this study are made publicly available as an open source toolset with graphical user interface

quality control is increasingly recognized as crucial aspect of mass spectrometry based proteomics several recent papers discuss relevant parameters for quality control and present applications to extract these from the instrumental raw data what has been missing however is standard data exchange format for reporting these performance metrics We therefore developed the qcML format an XML based standard that follows the design principles of the related mzML mzidentML mzquantML and traML standards from the HUPO PSI proteomics standards initiative In addition to the XML format we also provide tools for the calculation of wide range of quality metrics as well as database format and interconversion tools so that existing LIMS system can easily add relational storage of the quality control data to their existing schema We here describe the qcML specification along with possible use cases and an illustrative example of the subsequent analysis possibilities

protein phosphorylation is the best studied posttranslational modification and plays role in virtually every biological process phosphoproteomics is the analysis of protein phosphorylation on proteome wide scale and mainly uses the same instrumentation and analogous strategies as conventional mass spectrometry MS based proteomics measurements can be performed either in discovery type also known as shotgun mode or in targeted manner which monitors set of priori known phosphopeptides such as members of signal transduction pathway across biological samples here we delineate the different experimental levels at which measures can be taken to optimize the scope reliability and information content of phosphoproteomic analyses various chromatographic and chemical protocols exist to physically enrich phosphopeptides from proteolytic digests of biological samples subsequent mass spectrometric analysis revolves around peptide ion fragmentation to generate sequence information and identify the backbone sequence of phosphopeptides as well as the phosphate group attachment site and different modes of fragmentation like collision induced dissociation CID electron transfer dissociation ETD and higher energy collisional dissociation HCD have been established for phosphopeptide analysis computational tools are important for the identification and quantification of phosphopeptides and mapping of phosphorylation sites the deposition of large scale phosphoproteome datasets in public databases and the extraction of biologically meaningful information by data mining integration with other data types and descriptive or predictive modeling finally we discuss how orthogonal experimental approaches can be employed to validate newly identified phosphorylation sites on biochemical mechanistic and physiological level

chemical cross linking in combination with LC MS MS XL MS is an emerging technology to obtain low resolution structural distance restraints of protein and protein complexes these restraints can also be used to characterize protein complexes by integrative modeling of the XL MS data either in combination with other types of structural information or by themselves to establish spatial relationships of subunits in protein complexes here we present protocol that has been successfully used to generate XL MS data from multitude of native protein and protein complexes It includes the experimental steps for performing the cross linking reaction using disuccinimidyl suberate homobifunctional lysine reactive cross linking reagent the enrichment of cross linked peptide by peptide size exclusion chromatography SEC to remove smaller non cross linked peptide instructions for tandem MS analysis and the analysis of MS data via the open source computational software pipeline xquest and xprophet available from http proteomics ethz ch once established this robust protocol should take to complete and it is generally applicable to purified protein and protein complexes

adoption of targeted mass spectrometry MS approaches such as multiple reaction monitoring MRM to study biological and biomedical questions is well underway in the proteomics community successful application depends on the ability to generate reliable assay that uniquely and confidently identify target peptide in sample unfortunately there is wide range of criteria being applied to say that an assay has been successfully developed there is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated publications describing targeted MS assay for peptide frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs guidance must be developed so that targeted MS assay with established performance can be made widely distributed and applied by many labs worldwide To begin to address the problems and their solutions workshop was held at the national institutes of health with representatives from the multiple communities developing and employing targeted MS assay participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assay they develop work as intended and are achieving the required levels of performance using this fit for purpose approach the group defined three tiers of assay distinguished by their performance and extent of analytical characterization computational and statistical tools useful for the analysis of targeted MS results were described participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported this paper presents summary of the meeting and recommendations

targeted proteomics is method of choice for accurate and high throughput quantification of predefined sets of protein many workflows use isotope labeled reference peptide for every target protein which is time consuming and costly We report statistical approach for quantifying full protein panels with reduced set of reference peptide this label sparse approach achieves accurate quantification while reducing experimental cost and time It is implemented in the software tool sparsequant

stresses such as glucose depletion activate snf1 the saccharomyces cerevisiae ortholog of adenosine monophosphate activated protein kinase AMPK enabling adaptive cellular responses In addition to affecting transcription snf1 may also promote mRNA stability in gene specific manner To understand snf1 mediated signaling we used quantitative mass spectrometry to identify protein that were phosphorylated in snf1 dependent manner We identified snf1 dependent phosphopeptides in protein thirteen of these protein are involved inmRNA metabolism Of these we found that ccr4 the major cytoplasmic deadenylase dhh1 an RNA helicase and xrn1 an exoribonuclease were required for the glucose induced decay of snf1 dependent mRNAs that were activated by glucose depletion unexpectedly deletion of XRN1 reduced the accumulation of snf1 dependent transcripts that were synthesized during glucose depletion deletion of SNF1 rescued the synthetic lethality of simultaneous deletion of XRN1 and REG1 which encodes regulatory subunit of phosphatase that inhibits snf1 mutation of three snf1 dependent phosphorylation sites in xrn1 reduced glucose induced mRNA decay thus xrn1 is required for snf1 dependent mRNA homeostasis in response to nutrient availability

At the 12th annual HUPO world congress of proteomics in japan the human proteome project HPP presented scientific workshop sessions here we summarize highlights of ten workshops from the biology and disease driven HPP HPP teams and three from the HPP resource pillars highlights of the three chromosome centric HPP sessions appeared in the many articles of the HPP special issue of the journal of proteome research

T cell antigen receptor TCR mediated activation of cell requires the interaction of dozens of protein here we used quantitative mass spectrometry and activated primary CD4 + cell from mice in which tag for affinity purification was knocked into several gene to determine the composition and dynamics of multiprotein complexes that formed around the kinase zap70 and the adaptors lat and SLP most of the high confidence time resolved protein interactions we observed were previously unknown the surface receptor CD6 was able to initiate its own signaling pathway by recruiting SLP and the guanine nucleotide exchange factor vav1 regardless of the presence of lat our findings provide more complete model of TCR signaling in which CD6 constitutes signaling hub that contributes to the diversification of TCR signaling

enhanced transcription of the Rv2660c locus in response to starvation of mycobacterium tuberculosis H37Rv encouraged addition of the predicted Rv2660c protein to an improved vaccine formulation using strand specific RNA sequencing we show that the up regulated transcript is in fact small RNA encoded on the opposite strand to the annotated Rv2660c the transcript originates within prophage and is expressed only in strains that carry phiRv2 the small RNA contains both host and phage sequence and provides useful biomarker to monitor bacterial starvation during infection and or non replicating persistence using different approaches we do not find any evidence of Rv2660c at the level of mRNA or protein further efforts to understand the mechanism by which Rv2660c improves efficacy of the H56 vaccine are likely to provide insights into the pathology and immunology of tuberculosis

MiRNAs are post transcriptional regulators that contribute to the establishment and maintenance of gene expression patterns although their biogenesis and decay appear to be under complex control the implications of miRNA expression dynamics for the process that they regulate are not well understood We derived mathematical model of miRNA mediated gene regulation inferred its parameters from experimental data sets and found that the model describes well time dependent changes in mRNA protein and ribosome density levels measured upon miRNA transfection and induction the inferred parameters indicate that the timescale of miRNA dependent regulation is slower than initially thought delays in miRNA loading into argonaute protein and the slow decay of protein relative to mRNAs can explain the typically small changes in protein levels observed upon miRNA transfection for miRNAs to regulate protein expression on the timescale of day as miRNAs involved in cell cycle regulation do accelerated miRNA turnover is necessary

tissue homeostasis is controlled by signaling system that coordinate cell proliferation cell growth and cell shape upon changes in the cellular environment deregulation of these process is associated with human cancer and can occur at multiple levels of the underlying signaling system To gain an integrated view on signaling modules controlling tissue growth we analyzed the interaction proteome of the human hippo pathway an established growth regulatory signaling system the resulting high resolution network model of protein protein interactions among network components suggests participation of hippo pathway components in three distinct modules that all converge on the transcriptional co activator YAP1 one of the modules corresponds to the canonical hippo kinase cassette whereas the other two both contain hippo components in complexes with cell polarity protein quantitative proteomic data suggests that complex formation with cell polarity protein is dynamic and depends on the integrity of cell cell contacts collectively our systematic analysis greatly enhances our insights into the biochemical landscape underlying human hippo signaling and emphasizes multifaceted roles of cell polarity complexes in hippo mediated tissue growth control

protein complexes and protein interaction networks are essential mediators of most biological functions complexes supporting transient functions such as signal transduction process are frequently subject to dynamic remodeling currently the majority of studies on the composition of protein complexes are carried out by affinity purification and mass spectrometry AP MS and present static view of the system for better understanding of inherently dynamic biological process method to reliably quantify temporal changes of protein interaction networks are essential here we used affinity purification combined with sequential window acquisition of all theoretical spectra AP SWATH mass spectrometry to study the dynamics of the 3ß scaffold protein interactome after stimulation of the insulin PI3K AKT pathway the consistent and reproducible quantification of protein across all stimulation time points provided insights into the 3 interactome and its dynamic changes following IGF1 stimulation We therefore establish AP SWATH as tool to quantify dynamic changes in protein complex interaction networks

characterizing changes in protein protein interactions associated with sequence variants disease associated mutations or splice forms or following exposure to drugs growth factors or hormones is critical to understanding how protein complexes are built localized and regulated affinity purification AP coupled with mass spectrometry permits the analysis of protein interactions under near physiological conditions yet monitoring interaction changes requires the development of robust and sensitive quantitative approach especially for large scale studies in which cost and time are major considerations We have coupled AP to data independent mass spectrometric acquisition sequential window acquisition of all theoretical spectra SWATH and implemented an automated data extraction and statistical analysis pipeline to score modulated interactions We used AP SWATH to characterize changes in protein protein interactions imparted by the HSP90 inhibitor NVP AUY922 or melanoma associated mutations in the human kinase CDK4 We show that AP SWATH is robust label free approach to characterize such changes and propose scalable pipeline for system biology studies

deciphering physiological changes that mediate transition of mycobacterium tuberculosis between replicating and nonreplicating states is essential to understanding how the pathogen can persist in an individual host for decades We have combined RNA sequencing RNA seq of triphosphate enriched libraries with regular RNA seq to characterize the architecture and expression of tuberculosis promoters We identified over transcriptional start sites TSSs strikingly for of the gene with primary TSS the site of transcriptional initiation overlapped with the annotated start codon generating leaderless transcripts lacking UTR and hence the shine dalgarno sequence commonly used to initiate ribosomal engagement in eubacteria gene encoding protein with active growth functions were markedly depleted from the leaderless transcriptome and there was significant increase in the overall representation of leaderless mRNAs ina starvation model of growth arrest the high percentage of leaderless gene may have particular importance in the physiology of nonreplicating tuberculosis

among the wide range of proteomic technologies targeted mass spectrometry MS has shown great potential for biomarker studies To extend the degree of multiplexing achieved by selected reaction monitoring SRM we recently developed SWATH MS SWATH MS is variant of the emerging class of data independent acquisition DIA method and essentially converts the molecules in physical sample into perpetually re usable digital maps the thus generated SWATH maps are then mined using targeted data extraction strategy allowing us to profile disease related proteomes at high degree of reproducibility the successful application of both SRM and SWATH MS requires the priori generation of reference spectral maps that provide coordinates for quantification herein we demonstrate that the application of the mass spectrometric reference maps and the acquisition of personalized SWATH maps hold particular promise for accelerating the current process of biomarker discovery

microRNAs miRNAs guide argonaute ago protein to target mRNAs leading to gene silencing howeèer ago protein are not the actual mediators of gene silencing but interact with member of the GW182 protein family also known as GW protein which coordinates all downstream steps in gene silencing GW protein contain an terminal ago binding domain that is characterized by multiple GW repeats and terminal silencing domain with seèeral globular domains within the ago binding domain trp residue mediate the direct interaction with the ago protein here we haèe characterized the interaction of ago protein with GW protein in molecular detail using biochemical and NMR experiments we show that only subset of trp residue engage in ago interactions the trp residue are located in intrinsically disordered regions where flanking residue mediate additional weak interactions that might explain the importance of specific tryptophans using cross linking followed by mass spectrometry we map the GW protein interactions with ago2 which allows for structural modeling of ago GW182 interaction our data further indicate that the ago GW protein interaction might be two step process inèolèing the sequential binding of two tryptophans separated by spacer with minimal length of aa

chemical cross links identified by mass spectrometry generate distance restraints that reveal low resolution structural information on protein and protein complexes the technology to reliably generate such data has become mature and robust enough to shift the focus to the question of how these distance restraints can be best integrated into molecular modeling calculations here we introduce three workflows for incorporating distance restraints generated by chemical cross linking and mass spectrometry into ROSETTA protocols for comparative and de novo modeling and protein protein docking We demonstrate that the cross link validation and visualization software xwalk facilitates successful cross link data integration besides the protocols we introduce Xldb database of chemical cross links from different publications with intra protein and inter protein cross links where each cross link can be mapped on an experimental structure from the protein data bank finally we demonstrate on protein protein docking reference data set the impact of virtual cross links on protein docking calculations and show that an inter protein cross link can reduce on average the RMSD of docking prediction by the method and results presented here provide guidelines for the effective integration of chemical cross link data in molecular modeling calculations and should advance the structural analysis of particularly large and transient protein complexes via hybrid structural biology method

the ATP dependent chromatin remodeling complex SWR1 exchanges variant histone H2A H2B dimer for canonical H2A H2B dimer at nucleosomes flanking histone depleted regions such as promoters this localization of H2A is conserved throughout eukaryotes SWR1 is megadalton complex containing different polypeptides including the AAA+ ATpases rvb1 and rvb2 using electron microscopy we obtained the three dimensional structure of SWR1 and mapped its major functional components our data show that SWR1 contains single heterohexameric rvb1 rvb2 ring that together with the catalytic subunit swr1 brackets two independently assembled multisubunit modules We also show that SWR1 undergoes large conformational change upon engaging limited region of the nucleosome core particle our work suggests an important structural role for the rvbs and distinct substrate handling mode by SWR1 thereby providing structural framework for understanding the complex dimer exchange reaction

INO80 SWR1 family chromatin remodelers are complexes composed of subunits and molecular masses exceeding MDa their important role in transcription and genome maintenance is exchanging the histone variants H2A and H2A We report the architecture of cerevisiae INO80 using an integrative approach of electron microscopy crosslinking and mass spectrometry INO80 has an embryo shaped head neck body foot architecture and shows dynamic open and closed conformations We can assign an rvb1 rvb2 heterododecamer to the head in close contact with the ino80 snf2 domain ies2 and the arp5 module at the neck the high affinity nucleosome binding nhp10 module localizes to the body whereas the module that contains actin arp4 and arp8 maps to the foot structural and biochemical analyses indicate that the nucleosome is bound at the concave surface near the neck flanked by the rvb1 and arp8 modules our analysis establishes structural and functional framework for this family of large remodelers

targeted proteomics based on selected reaction monitoring SRM mass spectrometry is commonly used for accurate and reproducible quantification of protein analytes in complex biological mixtures strictly hypothesis driven SRM assay quantify each targeted protein by collecting measurements on its peptide fragment ions called transitions To achieve sensitive and accurate quantitative results experimental design and data analysis must consistently account for the variability of the quantified transitions this consistency is especially important in large experiments which increasingly require profiling up to hundreds of protein over hundreds of samples here we describe robust and automated workflow for the analysis of large quantitative SRM data sets that integrates data processing statistical protein identification and quantification and dissemination of the results the integrated workflow combines three software tools mprophet for peptide identification via probabilistic scoring SRmstats for protein significance analysis with linear mixed effect models and PASSEL public repository for storage retrieval and query of SRM data the input requirements for the protocol are files with SRM traces in mzXML format and file with list of transitions in text tab separated format the protocol is especially suited for data with heavy isotope labeled peptide internal standards We demonstrate the protocol on clinical data set in which the abundances of biomarker candidates were profiled in blood plasma samples of subjects with ovarian cancer or benign ovarian tumors the time frame to realize the protocol is weeks depending on the number of replicates used in the experiment

affinity purification coupled with mass spectrometry AP MS is widely used approach for the identification of protein protein interactions however for any given protein of interest determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants for example protein that interact with the solid phase support affinity reagent or epitope tag is challenging task the standard approach is to identify nonspecific interactions using one or more negative control purifications but many small scale AP MS studies do not capture complete accurate background protein set when available controls are limited fortunately negative controls are largely bait independent hence aggregating negative controls from multiple AP MS studies can increase coverage and improve the characterization of background associated with given experimental protocol here we present the contaminant repository for affinity purification the CRApome and describe its use for scoring protein protein interactions the repository currently available for homo sapiens and saccharomyces cerevisiae and computational tools are freely accessible at http www crapome org

the metabolic syndrome is collection of risk factors including obesity insulin resistance and hepatic steatosis which occur together and increase the risk of diseases such as diabetes cardiovascular disease and cancer In spite of intense research the complex etiology of insulin resistance and its association with the accumulation of triacylglycerides in the liver and with hepatic steatosis remains not completely understood here we performed quantitative measurements of protein involved in the insulin signaling pathway and central metabolism in liver homogenates of two genetically well defined mouse strains C57BL 6J and 129Sv that were subjected to sustained high fat diet We used targeted mass spectrometry by selected reaction monitoring SRM to generate accurate and reproducible quantitation of the targeted protein across different samples conditions and biological replicates generating one of the largest quantitative targeted proteomics data sets in mammalian tissues our results revealed rapid response to high fat diet that diverged early in the feeding regimen and evidenced response to high fat diet dominated by the activation of peroxisomal oxidation in C57BL 6J and by lipogenesis in 129Sv mice

background the phosphatase and tensin homolog PTEN tumor suppressor gene is deregulated in many advanced prostate cancers leading to activation of the phosphatidylinositol kinase PI3K akt mammalian target of rapamycin mTOR pathway and thus increased cell survival objective To evaluate everolimus an inhibitor of mTOR in patients with metastatic castration resistant prostate cancer mCRPC and to explore potentially predictive serum biomarkers by proteomics the significance of PTEN status in tumor tissue and the impact of everolimus on immune cell subpopulations and function design setting and participants total of chemotherapy naive patients with mCRPC and progressive disease were recruited to this single arm phase trial clinicaltrials gov identifier NCT00976755 intervention everolimus was administered continuously at dose of mg daily outcome measurements and statistical analysis the primary end point was progression free survival PFS at wk defined as the absence of prostate specific antigen PSA radiographic progression or clinical progression groups were compared using wilcoxon rank sum tests or fisher exact tests for continuous and discrete variables respectively time to event end points were analyzed using the kaplan meier method and univariate cox regression results and limitations total of patients confidence interval met the primary end point confirmed PSA response was seen in two and four further patients had PSA decline higher serum levels of carboxypeptidase and apolipoprotein were predictive for reaching the primary end point deletion of PTEN was associated with longer PFS and response treatment was associated with dose dependent decrease of CD3 CD4 and CD8 lymphocytes and CD8 proliferation and an increase in regulatory cell small sample size was the major limitation of the study conclusions everolimus activity in unselected patients with mCRPC is moderate but PTEN deletion could be predictive for response several serum glycoproteins were able to predict PFS at wk prospective validation of these potential biomarkers is warranted trial registration this study is registered with clinicaltrials gov with the identifier NCT00976755 results of this study were presented in part at the 47th annual meeting of the american society of clinical oncology june chicago IL USA and the annual meeting of the german austrian and swiss societies for oncology and hematology september october basel switzerland

We report high quality and system wide proteome catalogue covering protein of the predicted gene of fission yeast schizosaccharomyces pombe presenting the largest protein dataset to date for this important model organism We obtained this high proteome and peptide peptide protein coverage by combination of extensive sample fractionation high resolution orbitrap mass spectrometry and combined database searching using the iprophet software as part of the trans proteomics pipeline all raw and processed data are made accessible in the pombe peptideatlas the identified protein showed no biases in functional properties and allowed global estimation of protein abundances the high coverage of the peptideatlas allowed correlation with transcriptomic data in system wide manner indicating that post transcriptional process control the levels of at least half of all identified protein interestingly the correlation was not equally tight for all functional categories ranging from rs gt for protein involved in translation to rs lt for signal transduction protein moreover many protein involved in DNA damage repair could not be detected in the peptideatlas despite their high mRNA levels strengthening the translation on demand hypothesis for members of this protein class In summary the extensive and publicly available pombe peptideatlas together with the generated proteotypic peptide spectral library will be useful resource for future targeted in depth and quantitative proteomic studies on this microorganism

recent progress in genomic sequencing has revealed genotype phenotype information of enormous complexity and challenges earlier hypotheses on how phenotypes emerge from altered gene structures the field of proteomics has advanced in parallel and offers promising new concepts for modern interpretation of complex and nonlinear genotype phenotype relationships We are beginning to decipher global proteome organization with increasing throughput and accuracy these efforts revealed highly modular organization of the protein landscape here we discuss the challenges and implications emerging from modular protein landscape for better understanding of complex genotype phenotype patterns this journal is

research advancing our understanding of mycobacterium tuberculosis mtb biology and complex host mtb interactions requires consistent and precise quantitative measurements of mtb protein We describe the generation and validation of compendium of assay to quantify of the annotated mtb protein by the targeted mass spectrometric method selected reaction monitoring SRM furthermore we estimate the absolute abundance for of all mtb protein revealing dynamic range within the mtb proteome of over four orders of magnitude and identify previously unannotated protein As an example of the assay library utility we monitored the entire mtb dormancy survival regulon dosR which is linked to anaerobic survival and mtb persistence and show its dynamic protein level regulation during hypoxia In conclusion we present publicly available research resource that supports the sensitive precise and reproducible quantification of virtually any mtb protein by robust and widely accessible mass spectrometric method

the diauxic shift in saccharomyces cerevisiae is an ideal model to study how eukaryotic cell readjust their metabolism from glycolytic to gluconeogenic operation In this work we generated time resolved physiological data quantitative metabolome intracellular metabolites and proteome enzymes profiles We found that the diauxic shift is accomplished by three key events that are temporally organized reduction in the glycolytic flux and the production of storage compounds before glucose depletion mediated by downregulation of phosphofructokinase and pyruvate kinase reactions ii upon glucose exhaustion the reversion of carbon flow through glycolysis and onset of the glyoxylate cycle operation triggered by an increased expression of the enzymes that catalyze the malate synthase and cytosolic citrate synthase reactions and iii in the later stages of the adaptation the shutting down of the pentose phosphate pathway with change in NADPH regeneration moreover we identified the transcription factors associated with the observed changes in protein abundances taken together our results represent an important contribution toward system level understanding of how this adaptation is realized

cellular information processing via reversible protein phosphorylation requires tight control of the localization activity and substrate specificity of protein kinases which to large extent is accomplished by complex formation with other protein despite their critical role in cellular regulation and pathogenesis protein interaction information is available for only subset of the human protein kinases here we present global proteomic analysis of complexes of the human CMGC kinase group In addition to subgroup specific functional enrichment and modularity the identified high confidence kinase protein interactions provide specific biochemical context for many poorly studied CMGC kinases furthermore the analysis revealed kinase kinase subnetwork and candidate substrates for CMGC kinases finally the presented interaction proteome uncovered large set of interactions with protein genetically linked to range of human diseases including cancer suggesting additional routes for analyzing the role of CMGC kinases in controlling human disease pathways

expansion of functional islet cell mass is physiological process to compensate for increased insulin demand deficiency or pharmacological inhibition of the plasma membrane protease BACE2 enhances pancreatic cell function and proliferation and therefore BACE2 is putative target for the therapeutic intervention under conditions ofß cell loss and dysfunction To gain molecular understanding of BACE2 function we performed systematic and quantitative proteomic analysis to map the natural substrate repertoire of BACE2 and its homologue BACE1 in cell loss and gain of function studies of in vitro and in vivo models identified specific and functionally heterogeneous targets our analysis revealed non redundant roles of BACE1 in ectodomain shedding with BACE1 regulating broader and BACE2 more distinct set of cell enriched substrates including two protein of the seizure protein family SEZ6L and SEZ6L2 lastly our study provides insights into the global cell sheddome and secretome an important prerequisite to uncover novel mechanisms contributing to cell homeostasis and resource for therapeutic target and biomarker discoveries by the american society for biochemistry and molecular biology inc

We present computational pipeline for the quantification of peptide and protein in label free LC MS MS data sets the pipeline is composed of tools from the openMS software framework and is applicable to the processing of large experiments 50+ samples We describe several enhancements that we have introduced to openMS to realize the implementation of this pipeline they include new algorithms for centroiding of raw data for feature detection for the alignment of multiple related measurements and new tool for the calculation of peptide and protein abundances where possible we compare the performance of the new algorithms to that of their established counterparts in openMS We validate the pipeline on the basis of two small data sets that provide ground truths for the quantification there we also compare our results to those of maxquant and progenesis LC MS two popular alternatives for the analysis of label free data We then show how our software can be applied to large heterogeneous data set of LC MS MS runs

the characterization of all protein complexes of human cell under defined physiological conditions using affinity purification mass spectrometry AP MS is highly desirable step in the quest to understand the phenotypic effects of genomic information however such challenging goal has not yet been achieved as it requires reproducibility of the experimental workflow and high data consistency across different studies and laboratories We systematically investigated the reproducibility of standardized AP MS workflow by performing rigorous interlaboratory comparative analysis of the interactomes of human kinases We show that it is possible to achieve high interlaboratory reproducibility of this standardized workflow despite differences in mass spectrometry configurations and subtle sample preparation related variations and that combination of independent data sets improves the approach sensitivity resulting in even more detailed networks our analysis demonstrates the feasibility of obtaining high quality map of the human protein interactome with multilaboratory project

protein biomarkers have the potential to transform medicine as they are clinically used to diagnose diseases stratify patients and follow disease states even though large number of potential biomarkers have been proposed over the past few years almost none of them have been implemented so far in the clinic one of the reasons for this limited success is the lack of technologies to validate proposed biomarker candidates in larger patient cohorts this limitation could be alleviated by the use of antibody independent validation method such as selected reaction monitoring SRM similar to measurements based on affinity reagents SRM based targeted mass spectrometry also requires the generation of definitive assay for each targeted analyte here we present library of SRM assay for glycosites enabling the multiplexed evaluation of clinically relevant glycoproteins as biomarker candidates We demonstrate that this resource can be utilized to select SRM assay sets for cancer associated glycoproteins for their subsequent multiplexed and consistent quantification in human plasma samples We show that glycoproteins spanning orders of magnitude in abundance can be quantified and that previously reported abundance differences in various cancer types can be recapitulated together the established glycoprotein SRMatlas resource facilitates parallel efficient consistent and sensitive evaluation of proposed biomarker candidates in large clinical sample cohorts

virtually all the biological process are controlled and catalyzed by protein which are in many cases in complexes with other protein therefore understanding the architecture and structure of protein complexes is critical to understanding their biological role and function traditionally high resolution data for structural analysis of protein or protein complexes have been generated by the powerful method of ray crystallography and nuclear magnetic resonance NMR spectroscopy more recently mass spectrometry MS based method have been developed that provide low resolution structural information which contributes to the determination of the native structure of protein complexes that have remained refractory to the high resolution method native MS and affinity purification coupled with MS AP MS have been used to characterize the composition stoichiometry and connectivity of protein complexes chemical cross linking MS CX MS provides protein protein interaction data supplemented with distance information that indicates residue that are in close spatial proximity in the native protein structure hydrogen deuterium exchange combined with MS has been used to map protein protein binding sites here we focus on recent developments in CX MS and native MS and their application to challenging problems in structural biology

SWATH MS is data independent acquisition method that generates in single measurement complete recording of the fragment ion spectra of all the analytes in biological sample for which the precursor ions are within predetermined versus retention time window To assess the performance and suitability of SWATH MS based protein quantification for clinical use we compared SWATH MS and SRM MS based quantification of linked glycoproteins in human plasma commonly used sample for biomarker discovery using dilution series of isotopically labeled heavy peptide representing biomarker candidates the LOQ of SWATH MS was determined to reach fmol at peptide level by targeted data analysis which corresponds to concentration of ng protein mL in plasma while SRM reached peptide LOQ of fmol moreover the quantification of endogenous glycoproteins using SWATH MS showed high degree of reproducibility with the mean CV of correlating well with SRM results R2 overall SWATH MS measurements showed slightly lower sensitivity and comparable reproducibility to state of the art SRM measurements for targeted quantification of the glycosites in human blood however significantly larger number of peptide can be quantified per analysis We suggest that SWATH MS analysis combined with glycoproteome enrichment in plasma samples is promising integrative proteomic approach for biomarker discovery and verification

circulating levels of insulin and glucagon reflect the nutritional state of animals and elicit regulatory responses in the liver that maintain glucose and lipid homeostasis the transcription factor foxa2 activates lipid metabolism and ketogenesis during fasting and is inhibited via insulin PI3K akt signaling mediated phosphorylation at thr156 and nuclear exclusion here we show that in addition foxa2 is acetylated at the conserved residue lys259 following inhibition of histone deacetylases HDACs class III and the cofactors p300 and sirT1 are involved in foxa2 acetylation and deacetylation respectively physiologically fasting states and glucagon stimulation are sufficient to induce foxa2 acetylation introduction of the acetylation mimicking K259Q or deficient K259R mutations promotes or inhibits foxa2 activity respectively and adenoviral expression of foxa2 K259Q augments expression of gene involved in fatty acid oxidation and ketogenesis our study reveals molecular mechanism by which glucagon signaling activates fasting response through acetylation of foxa2

topoisomerase TOP1 inhibitors are an important class of anticancer drugs the cytotoxicity of TOP1 inhibitors can be modulated by replication fork reversal through process that requires poly ADP ribose polymerase PARP activity whether regressed forks can efficiently restart and what factors are required to restart fork progression after fork reversal are still unknown We have combined biochemical and EM approaches with single molecule DNA fiber analysis to identify key role for human RECQ1 helicase in replication fork restart after TOP1 inhibition that is not shared by other human recQ protein We show that the poly ADP ribosyl ation activity of PARP1 stabilizes forks in the regressed state by limiting their restart by RECQ1 these studies provide new mechanistic insights into the roles of RECQ1 and PARP in DNA replication and offer molecular perspectives to potentiate chemotherapeutic regimens based on TOP1 inhibition copyright

regulation of gene expression involves the orchestrated interaction of large number of protein with transcriptional regulatory elements in the context of chromatin our understanding of gene regulation is limited by the lack of protein measurement technology that can systematically detect and quantify the ensemble of protein associated with the transcriptional regulatory elements of specific gene here we introduce set of selected reaction monitoring SRM assay for the systematic measurement of protein with known or suspected roles in transcriptional regulation at RNA polymerase II transcribed promoters in saccharomyces cerevisiae measurement of these protein in nuclear extracts by SRmpermitted the reproducible quantification of of the protein over wide range of abundances By deploying the assay to systematically identify DNA binding transcriptional regulators that interact with the environmentally regulated FLO11 promoter in cell extracts we identified regulators that bound specifically to distinct regions along bp of the regulatory sequence importantly the dataset includes number of regulators that have been shown to either control FLO11 expression or localize to these regulatory regions in vivo We further validated the utility of the approach by demonstrating that two of the SRM identified factors mot3 and azf1 are required for proper FLO11 expression these results demonstrate the utility of SRM based targeted proteomics to guide the identification of gene specific transcriptional regulators

cytosolic compartmentalization through liquid liquid unmixing such as the formation of RNA granules is involved in many cellular process and might be used to regulate signal transduction however specific molecular mechanisms by which liquid liquid unmixing and signal transduction are coupled remain unknown here we show that during cellular stress the dual specificity kinase DYRK3 regulates the stability of granule like structures and mTORC1 signaling DYRK3 displays cyclic partitioning mechanism between stress granules and the cytosol via low complexity domain in its terminus and its kinase activity when DYRK3 is inactive it prevents stress granule dissolution and the release of sequestered mTORC1 when DYRK3 is active it allows stress granule dissolution releasing mTORC1 for signaling and promoting its activity by directly phosphorylating the mTORC1 inhibitor PRAS40 this mechanism links cytoplasmic compartmentalization via liquid phase transitions with cellular signaling

experience from different fields of life sciences suggests that accessible complete reference maps of the components of the system under study are highly beneficial research tools examples of such maps include libraries of the spectroscopic properties of molecules or databases of drug structures in analytical or forensic chemistry such maps and method to navigate them constitute reliable assay to probe any sample for the presence and amount of molecules contained in the map So far attempts to generate such maps for any proteome have failed to reach complete proteome coverage here we use strategy based on high throughput peptide synthesis and mass spectrometry to generate an almost complete reference map of the genome predicted protein of the saccharomyces cerevisiae proteome We generated two versions of this mass spectrometric map one supporting discovery driven shotgun and the other supporting hypothesis driven targeted proteomic measurements together the two versions of the map constitute complete set of proteomic assay to support most studies performed with contemporary proteomic technologies To show the utility of the maps we applied them to protein quantitative trait locus QTL analysis which requires precise measurement of the same set of peptide over large number of samples protein measurements over cerevisiae strains revealed complex relationship between independent genetic loci influencing the levels of related protein our results suggest that selective pressure favours the acquisition of sets of polymorphisms that adapt protein levels but also maintain the stoichiometry of functionally related pathway members

pyrococcus furiosus pfu is an excellent organism to generate reference samples for proteomics laboratories because of its moderately sized genome and very little sequence duplication within the genome We demonstrated stable and consistent method to prepare protein in bulk that eliminates growth and preparation as source of uncertainty in the standard We performed several proteomic studies in different laboratories using each laboratory specific workflow as well as separate and integrated data analysis this study demonstrated that pfu whole cell lysate provides suitable protein sample complexity to not only validate proteomic method work flows and benchmark new instruments but also to facilitate comparison of experimental data generated over time and across instruments or laboratories

the TORC1 and PKA protein kinases are central elements of signaling networks that regulate eukaryotic cell proliferation in response to growth factors and or nutrients In yeast attenuation of signaling by these kinases following nitrogen and or carbon limitation activates the protein kinase rim15 which orchestrates the initiation of reversible cellular quiescence program to ensure normal chronological life span the molecular elements linking rim15 to distal readouts including the expression of msn2 and gis1 dependent gene involve the endosulfines igo1 here we show that rim15 analogous to the greatwall kinase in xenopus phosphorylates endosulfines to directly inhibit the cdc55 protein phosphatase 2A PP2Acdc55 inhibition of PP2Acdc55 preserves gis1 in phosphorylated state and consequently promotes its recruitment to and activation of transcription from promoters of specific nutrient regulated gene these results close gap in our perception of and delineate role for PP2Acdc55 in TORC1 PKA mediated regulation of quiescence and chronological life span

the biology and disease oriented branch of the human proteome project HPP was established by the human proteome organization HUPO with the main goal of supporting the broad application of state of theart measurements of protein and proteomes by life scientists studying the molecular mechanisms of biological process and human disease this will be accomplished through the generation of research and informational resources that will support the routine and definitive measurement of the process or disease relevant protein the HPP is highly complementary to the HPP and will provide datasets and biological characterization useful to the HPP teams In this manuscript we describe the goals the plans and the current status of the of the HPP

background serum biomarkers can improve diagnosis and treatment of malignant pleural mesothelioma MPM however the evaluation of potential new serum biomarker candidates is hampered by lack of assay technologies for their clinical evaluation here we followed hypothesis driven targeted proteomics strategy for the identification and clinical evaluation of MPM candidate biomarkers in serum of patient cohorts results based on the hypothesis that cell surface exposed glycoproteins are prone to be released from tumor cell to the circulatory system we screened the surfaceome of model cell lines for potential MPM candidate biomarkers selected reaction monitoring SRM assay technology allowed for the direct evaluation of the newly identified candidates in serum our evaluation of candidate biomarkers in the context of training and an independent validation set revealed reproducible glycopeptide signature of MPM in serum which complemented the MPM biomarker mesothelin conclusions our study shows that SRM assay technology enables the direct clinical evaluation of protein derived candidate biomarker panels for which clinically reliable ELISA currently do not exist

liquid chromatography coupled with tandem mass spectrometry LC MS MS is widely used for quantitative proteomic investigations the typical output of such studies is list of identified and quantified peptide the biological and clinical interest is however usually focused on quantitative conclusions at the protein level furthermore many investigations ask complex biological questions by studying multiple interrelated experimental conditions therefore there is need in the field for generic statistical models to quantify protein levels even in complex study designs We propose general statistical modeling approach for protein quantification in arbitrary complex experimental designs such as time course studies or those involving multiple experimental factors the approach summarizes the quantitative experimental information from all the features and all the conditions that pertain to protein It enables both protein significance analysis between conditions and protein quantification in individual samples or conditions We implement the approach in an open source based software package Msstats suitable for researchers with limited statistics and programming background We demonstrate using as examples two experimental investigations with complex designs that simultaneous statistical modeling of all the relevant features and conditions yields higher sensitivity of protein significance analysis and higher accuracy of protein quantification as compared to commonly employed alternatives the software is available at http www stat purdue edu ovitek software html

selected reaction monitoring mass spectrometry SRM MS is targeted proteomics technology used to identify and quantify protein with high sensitivity specificity and high reproducibility execution of SRM MS relies on protein specific SRM assay set of experimental parameters that requires considerable effort to develop here we present proteome wide SRM assay repository for the gram positive human pathogen group streptococcus using multi layered approach we generated SRM assay for distinct group streptococcus peptide followed by extensive testing of the selected reaction monitoring assay in different group streptococcus protein pools based on the number of SRM assay observations we created rule based selected reaction monitoring assay scoring model to select the most suitable assay per protein for given cellular compartment and bacterial state the resource described here represents an important tool for deciphering the group streptococcus proteome using selected reaction monitoring and we anticipate that concepts described here can be extended to other pathogens

As frequent post translational modification protein phosphorylation regulates many cellular process although several hundred phosphorylation sites have been mapped to metabolic enzymes in saccharomyces cerevisiae functionality was demonstrated for few of them here we describe novel approach to identify in vivo functionality of enzyme phosphorylation by combining flux analysis with proteomics and phosphoproteomics focusing on the network of enzymes that constitute the yeast central carbon and amino-acid metabolism we combined protein and phosphoprotein levels to identify enzymes that change their degree of phosphorylation during growth under five conditions correlations between previously determined intracellular fluxes and phosphoprotein abundances provided first functional evidence for five novel phosphoregulated enzymes in this network adding to nine known phosphoenzymes for the pyruvate dehydrogenase complex E1 subunit pda1 and the newly identified phosphoregulated glycerol phosphate dehydrogenase gpd1 and phosphofructose kinase complex subunit pfk2 we then validated functionality of specific phosphosites through absolute peptide quantification by targeted mass spectrometry metabolomics and physiological flux analysis in mutants with genetically removed phosphosites these results demonstrate the role of phosphorylation in controlling the metabolic flux realised by these three enzymes

transcription initiation by eukaryotic RNA polymerase pol III relies on the TFIIE related subcomplex C82 here we combine cross linking and hydroxyl radical probing to position the C82 subcomplex around the pol III active center cleft the extended winged helix WH domains and of C82 localize to the polymerase domains clamp head and clamp core respectively and the two WH domains of C34 span the polymerase cleft from the coiled coil region of the clamp to the protrusion the WH domains of C82 and C34 apparently cooperate with other mobile regions flanking the cleft during promoter DNA binding opening and loading together with published data our results complete the subunit architecture of pol III and indicate that all TFIIE related components of eukaryotic and archaeal transcription system adopt an evolutionarily conserved location in the upper part of the cleft that supports their functions in open promoter complex formation and stabilization

the complete and specific proteolytic cleavage of protein samples into peptide is crucial for the success of every shotgun LC MS MS experiment In particular popular peptide based label free and targeted mass spectrometry approaches rely on efficient generation of fully cleaved peptide to ensure accurate and sensitive protein quantification In contrast to previous studies we globally and quantitatively assessed the efficiency of different digestion strategies using yeast cell lysate label free quantification and statistical analysis digestion conditions include double tryptic surfactant assisted and tandem combinatorial lys trypsin digestion In comparison to tryptic digests lys trypsin digests were found most efficient to yield fully cleaved peptide while reducing the abundance of miscleaved peptide subsequent sequence context analysis revealed improved digestion performances of lys trypsin for miscleaved sequence stretches flanked by charged basic and particulary acidic residue furthermore targeted MS analysis demonstrated more comprehensive protein cleavage only after lys trypsin digestion resulting in more accurrate absolute protein quantification and extending the number of peptide suitable for SRM assay development therefore we conclude that serial lys trypsin digestion is highly attractive for most applications in quantitative MS based proteomics building on in solution digestion schemes

polycomb repressive complex PRC2 is essential for gene silencing establishing transcriptional repression of specific gene by tri methylating lysine of histone H3 process mediated by cofactors such as AEBP2 In spite of its biological importance little is known about PRC2 architecture and subunit organization here we present the first three dimensional electron microscopy structure of the human PRC2 complex bound to its cofactor AEBP2 using novel internal protein tagging method in combination with isotopic chemical cross linking and mass spectrometry we have localized all the PRC2 subunits and their functional domains and generated detailed map of interactions the position and stabilization effect of AEBP2 suggests an allosteric role of this cofactor in regulating gene silencing regions in PRC2 that interact with modified histone tails are localized near the methyltransferase site suggesting molecular mechanism for the chromatin based regulation of PRC2 activity

data on absolute molecule numbers will empower the modeling understanding and comparison of cellular functions and biological system We quantified transcriptomes and proteomes in fission yeast during cellular proliferation and quiescence this rich resource provides the first comprehensive reference for all RNA and most protein concentrations in eukaryote under two key physiological conditions the integrated data set supports quantitative biology and affords unique insights into cell regulation although mRNAs are typically expressed in narrow range above copy cell most long noncoding RNAs except for distinct subset are tightly repressed below copy cell cell cycle regulated transcription tunes mRNA numbers to phase specific requirements but can also bring about more switch like expression protein greatly exceed mRNAs in abundance and dynamic range and concentrations are regulated to functional demands upon transition to quiescence the proteome changes substantially but in stark contrast to mRNAs protein do not uniformly decrease but scale with cell volume

glioblastoma is the most common primary brain tumor in adults with low average survival time after diagnosis In order to improve glioblastoma treatment new drug accessible targets need to be identified cell surface glycoproteins are prime drug targets due to their accessibility at the surface of cancer cell To overcome the limited availability of suitable antibodies for cell surface protein detection we performed comprehensive mass spectrometric investigation of the glioblastoma surfaceome our combined cell surface capturing analysis of primary ex vivo glioblastoma cell lines in combination with established glioblastoma cell lines revealed glycoproteins which vastly extends the known data of surfaceome drug targets at subcellular resolution We provide direct evidence of common glioblastoma cell surface glycoproteins and an approximate estimate of their abundances information that could not be derived from genomic and or transcriptomic glioblastoma studies apart from our pharmaceutically valuable repertoire of already and potentially drug accessible cell surface glycoproteins we built mass spectrometry based toolbox enabling directed sensitive and repetitive glycoprotein measurements for clinical follow up studies the included skyline glioblastoma SRM assay library provides an elevated starting point for parallel testing of the abundance level of the detected glioblastoma surfaceome members in future drug perturbation experiments

many cellular responses are triggered by protein drugs or pathogens binding to cell surface receptors but it can be challenging to identify which receptors are bound by given ligand here we describe TRICEPS chemoproteomic reagent with three moieties one that binds ligands containing an amino group second that binds glycosylated receptors on living cell and biotin tag for purifying the receptor peptide for identification by quantitative mass spectrometry We validated this ligand based receptor capture LRC technology using insulin transferrin apelin epidermal growth factor the therapeutic antibody trastuzumab and two DARpins targeting erbB2 In some cases we could also determine the approximate ligand binding sites on the receptors using TRICEPS to label intact mature vaccinia viruses we identified the cell surface protein AXL M6PR DAG1 CSPG4 and CDH13 as binding factors on human cell this technology enables the identification of receptors for many types of ligands under near physiological conditions and without the need for genetic manipulations

background the breast and ovarian cancer suppressor BRCA1 is essential for cellular responses to DNA damage It heterodimerizes with BARD1 to acquire an E3 ubiquitin Ub ligase activity that is often compromised by cancer associated mutations neither the significance of this activity to damage responses nor relevant in vivo substrate is clear results We have separated DNA damage responses requiring the BRCA1 E3 ligase from those independent of it using gene targeted point mutation in vertebrate DT40 cell that abrogates BRCA1 catalytic activity without perturbing BARD1 binding We show that BRCA1 ubiquitylates claspin an essential coactivator of the CHK1 checkpoint kinase after topoisomerase inhibition but not DNA crosslinking by mitomycin BRCA1 E3 inactivation decreases chromatin bound claspin levels and impairs homology directed DNA repair by interrupting signal transduction from the damage activated ATR kinase to its effector CHK1 conclusions our findings identify claspin as an in vivo substrate for the BRCA1 E3 ligase and suggest that its modification selectively triggers CHK1 activation for the homology directed repair of subset of genotoxic lesions this mechanism unexpectedly defines an essential but selective function for BRCA1 E3 ligase activity in cellular responses to DNA damage

the identification of proximate amino-acid by chemical cross linking and mass spectrometry XL MS facilitates the structural analysis of homogeneous protein complexes We gained distance restraints on modular interaction network of protein complexes affinity purified from human cell by applying an adapted XL MS protocol systematic analysis of human protein phosphatase 2A PP2A complexes identified interprotein and intraprotein cross links that link specific trimeric PP2A complexes to multitude of adaptor protein that control their cellular functions spatial restraints guided molecular modeling of the binding interface between immunoglobulin binding protein IGBP1 and PP2A and revealed the topology of TCP1 ring complex TRiC chaperonin interacting with the PP2A regulatory subunit 2ABG this study establishes XL MS as an integral part of hybrid structural biology approaches for the analysis of endogenous protein complexes

this white paper sets out life sciences grand challenge for proteomics technologies to enhance our understanding of complex biological system link genomes with phenotypes and bring broad benefits to the biosciences and the US economy the paper is based on workshop hosted by the national institute of standards and technology NIST in gaithersburg MD february with participants from many federal agencies and research communities under the aegis of the US national science and technology council NSTC opportunities are identified for coordinated effort to achieve major technology based goals and address societal challenges in health agriculture nutrition energy environment national security and economic development

proteome information resources of farm animals are lagging behind those of the classical model organisms despite their important biological and economic relevance here we present bovine peptideatlas representing first collection of bos taurus proteome data sets within the peptideatlas framework this database was built primarily as source of information for designing selected reaction monitoring assay for studying milk production and mammary gland health but it has an intrinsic general value for the farm animal research community the bovine peptideatlas comprises protein at false discovery rate FDR and distinct peptide at FDR identified in samples from six tissues the peptideatlas web interface has rich set of visualization and data exploration tools enabling users to interactively mine information about individual protein and peptide their prototypic features genome mappings and supporting spectral evidence

the mass spectrometric identification of chemically cross linked peptide CXMS specifies spatial restraints of protein complexes these values complement data obtained from common structure determination techniques generic method for determining false discovery rates of cross linked peptide assignments are currently lacking thus making data sets from CXMS studies inherently incomparable here we describe an automated target decoy strategy and the software tool xprophet which solve this problem for large multicomponent protein complexes

knowledge of the protein networks interacting with the amyloid precursor protein APP in vivo can shed light on the physiological function of APP To date most protein interacting with the APP intracellular domain AICD have been identified by yeast two hybrid screens which only detect direct interaction partners We used proteomics based approach by biochemically isolating tagged APP from the brains of transgenic mice and subjecting the affinity purified complex to mass spectrometric MS analysis using two different quantitative MS approaches we compared the protein composition of affinity purified samples isolated from wild type mice versus transgenic mice expressing tagged APP this enabled us to assess truly enriched protein in the transgenic sample and yielded an overlapping set of protein containing the major protein involved in synaptic vesicle endo and exocytosis confocal microscopy analyses of cotransfected primary neurons showed colocalization of APP with synaptic vesicle protein in vesicular structures throughout the neurites We analyzed the interaction of APP with these protein using pulldown experiments from transgenic mice or cotransfected cell followed by western blotting synaptotagmin stg1 resident synaptic vesicle protein was found to directly bind to APP We fused citrine and cerulean to APP and the candidate protein and measured fluorescence resonance energy transfer FRET in differentiated SH SY5Y cell differentially tagged APPs showed clear sensitized FRET emission in line with the described dimerization of APP among the candidate APP interacting protein again only stg1 was in close proximity to APP our results strongly argue for function of APP in synaptic vesicle turnover in vivo thus in addition to the APP cleavage product Aß which influences synaptic transmission at the postsynapse APP interacts with the calcium sensor of synaptic vesicles and might thus play role in the regulation of synaptic vesicle exocytosis

selected reaction monitoring SRM also called multiple reaction monitoring has become an invaluable tool for targeted quantitative proteomic analyses but its application can be compromised by nonoptimal selection of transitions In particular complex backgrounds may cause ambiguities in SRM measurement results because peptide with interfering transitions similar to those of the target peptide may be present in the sample here we developed computer program the SRMcollider that calculates nonredundant theoretical SRM assay also known as unique ion signatures UIS for given proteomic background We show theoretically that UIS of three transitions suffice to conclusively identify of all yeast peptide and of all human peptide using predicted retention times the SRMcollider also simulates time scheduled SRM acquisition which reduces the number of interferences to consider and leads to fewer transitions necessary to construct an assay By integrating experimental fragment ion intensities from large scale proteome synthesis efforts SRMatlas with the information content based UIS we combine two orthogonal approaches to create high quality SRM assay ready to be deployed We provide user friendly open source implementation of an algorithm to calculate UIS of any order that can be accessed online at http www srmcollider org to find interfering transitions finally our tool can also simulate the specificity of novel data independent MS acquisition method in Q1 Q3 space this allows us to predict parameters for these method that deliver specificity comparable with that of SRM using SRM interference information in addition to other sources of information can increase the confidence in an SRM measurement We expect that the consideration of information content will become standard step in SRM assay design and analysis facilitated by the SRMcollider

mass spectrometry MS based proteomics has significantly contributed to the development of system biology new paradigm for the life sciences in which biological process are addressed in terms of dynamic networks of interacting molecules because of its advanced analytical capabilities MS based proteomics has been used extensively to identify the components of biological system and it is the method of choice to consistently quantify the effects of network perturbation in time and space herein we review recent contributions of MS to system biology and discuss several examples that illustrate the importance of mass spectrometry to elucidate the components and interactions of molecular networks

the rigorous testing of hypotheses on suitable sample cohorts is major limitation in translational research this is particularly the case for the validation of protein biomarkers the lack of accurate reproducible and sensitive assay for most protein has precluded the systematic assessment of hundreds of potential marker protein described in the literature here we describe high throughput method for the development and refinement of selected reaction monitoring SRM assay for human protein the method was applied to generate such assay for more than cancer associated protein which are functionally related to candidate cancer driver mutations We used the assay to determine the detectability of the target protein in two clinically relevant samples plasma and urine one hundred eighty two protein were detected in depleted plasma spanning five orders of magnitude in abundance and reaching below concentration of ng ml the narrower concentration range of protein in urine allowed the detection of protein moreover we demonstrate that these SRM assay allow reproducible quantification by monitoring biomarker candidates across patient plasma samples through public access to the entire assay library researchers will be able to target their cancer associated protein of interest in any sample type using the detectability information in plasma and urine as guide the generated expandable reference map of SRM assay for cancer associated protein will be valuable resource for accelerating and planning biomarker verification studies

microRNAs miRNAs are small noncoding RNAs that negatively regulate gene expression As miRNAs are involved in wide range of biological process and diseases much effort has been invested in identifying their mRNA targets here we present novel combinatorial approach RIP chip SRM RNA binding protein immunopurification + microarray + targeted protein quantification via selected reaction monitoring to identify de novo high confidence miRNA targets in the nematode caenorhabditis elegans We used differential RIP chip analysis of miRNA induced silencing complexes from wild type and miRNA mutant animals followed by quantitative targeted proteomics via selected reaction monitoring to identify and validate mRNA targets of the elegans bantam homolog miR comparison of total mRNA and protein abundance changes in mir mutant and wild type animals indicated that the direct bantam miR targets identified here are mainly regulated at the level of protein abundance not mRNA stability

eicosanoids constitute diverse class of bioactive lipid mediators that are produced from arachidonic acid and play critical roles in cell signaling and inflammatory aspects of numerous diseases We have previously quantified eicosanoid metabolite production in RAW264 macrophage cell in response to toll like receptor signaling and analyzed the levels of transcripts coding for the enzymes involved in the eicosanoid metabolite biosynthetic pathways We now report the quantification of changes in protein levels under similar experimental conditions in RAW264 macrophages by multiple reaction monitoring mass spectrometry an accurate targeted protein quantification method the data complete the first fully integrated genomic proteomic and metabolomic analysis of the eicosanoid biochemical pathway

In the life sciences new paradigm is emerging that places networks of interacting molecules between genotype and phenotype these networks are dynamically modulated by multitude of factors and the properties emerging from the network as whole determine observable phenotypes this paradigm is usually referred to as system biology network biology or integrative biology mass spectrometry MS based proteomics is central life science technology that has realized great progress toward the identification quantification and characterization of the protein that constitute proteome here we review how MS based proteomics has been applied to network biology to identify the nodes and edges of biological networks to detect and quantify perturbation induced network changes and to correlate dynamic network rewiring with the cellular phenotype We discuss future directions for MS based proteomics within the network biology paradigm

RNA polymerase pol contains subunit catalytic core that is related to the core of pol II and includes subunit A12 In addition pol contains the heterodimeric subcomplexes A14 and A49 which are related to the pol II subcomplex rpb4 and the pol II initiation factor TFIIF respectively here we used lysine lysine crosslinking mass spectrometry MS and modeling based on five crystal structures to extend the previous homology model of the pol core to confirm the location of A14 and to position A12 and A49 on the core In the resulting model of pol the terminal ribbon ribbon domain of A12 reaches the active site via the polymerase pore like the ribbon of the pol II cleavage factor TFIIS explaining why the intrinsic RNA cleavage activity of pol is strong in contrast to the weak cleavage activity of pol II the A49 dimerization module resides on the polymerase lobe like TFIIF whereas the A49 tWH domain resides above the cleft resembling parts of TFIIE this indicates that pol and also pol III are distantly related to pol II TFIIS TFIIF TFIIE complex

eicosanoids constitute diverse class of bioactive lipid mediators that are produced from arachidonic acid and play critical roles in cell signaling and inflammatory aspects of numerous diseases We have previously quantified eicosanoid metabolite production in RAW264 macrophage cell in response to toll like receptor signaling and analyzed the levels of transcripts coding for the enzymes involved in the eicosanoid metabolite biosynthetic pathways We now report the quantification of changes in protein levels under similar experimental conditions in RAW264 macrophages by multiple reaction monitoring mass spectrometry an accurate targeted protein quantification method the data complete the first fully integrated genomic proteomic and metabolomic analysis of the eicosanoid biochemical pathway

due to the enormous complexity of proteomes which constitute the entirety of protein species expressed by certain cell or tissue proteome wide studies performed in discovery mode are still limited in their ability to reproducibly identify and quantify all protein present in complex biological samples therefore the targeted analysis of informative subsets of the proteome has been beneficial to generate reproducible data sets across multiple samples here we review the repertoire of antibody and mass spectrometry MS based analytical tools which is currently available for the directed analysis of predefined sets of protein the topics of emphasis for this review are selected reaction monitoring SRM mass spectrometry emerging tools to control error rates in targeted proteomic experiments and some representative examples of applications the ability to cost and time efficiently generate specific and quantitative assay for large numbers of protein and posttranslational modifications has the potential to greatly expand the range of targeted proteomic coverage in biological studies this article is part of special section entitled understanding genome regulation and genetic diversity by mass spectrometry

subversion of host organism cAMP signaling is an efficient and widespread mechanism of microbial pathogenesis bartonella effector proteinA bepA of vasculotumorigenic bartonella henselae protects the infected human endothelial cell against apoptotic stimuli by elevation of cellular cAMP levels by an as yet unknown mechanism here adenylyl cyclase AC and the subunit of the AC stimulating protein gas were identified as potential cellular target protein for bepA by gel free proteomics results of the proteomics screen were evaluated for physical and functional interaction by heterologous in vivo coexpression system where human AC activity was reconstituted under the regulation of gas and bepA in escherichia coli ii in vitro AC assay with membrane anchored fulllength human AC and recombinant bepA and gas iii surface plasmon resonance experiments and iv an in vivo fluorescence bimolecular complementation analysis the data demonstrate that bepA directly binds host cell AC to potentiate the gas dependent cAMP production As opposed to the known microbial mechanisms such as ADP ribosylation of protein subunits by cholera and pertussis toxins the fundamentally different bepA mediated elevation of host cell cAMP concentration appears subtle and is dependent on the stimulus of protein coupled receptor released gas We propose that this mechanism contributes to the persistence of bartonella henselae in the chronically infected vascular endothelium

most proteomic studies use liquid chromatography coupled to tandem mass spectrometry to identify and quantify the peptide generated by the proteolysis of biological sample however with the current method it remains challenging to rapidly consistently reproducibly accurately and sensitively detect and quantify large fractions of proteomes across multiple samples here we present new strategy that systematically queries sample sets for the presence and quantity of essentially any protein of interest It consists of using the information available in fragment ion spectral libraries to mine the complete fragment ion maps generated using data independent acquisition method for this study the data were acquired on fast high resolution quadrupole quadrupole time of flight TOF instrument by repeatedly cycling through consecutive Da precursor isolation windows swaths this SWATH MS acquisition setup generates in single sample injection time resolved fragment ion spectra for all the analytes detectable within the precursor range and the user defined retention time window We show that suitable combinations of fragment ions extracted from these data sets are sufficiently specific to confidently identify query peptide over dynamic range of orders of magnitude even if the precursors of the queried peptide are not detectable in the survey scans We also show that queried peptide are quantified with consistency and accuracy comparable with that of selected reaction monitoring the gold standard proteomic quantification method moreover targeted data extraction enables ad libitum quantification refinement and dynamic extension of protein probing by iterative re mining of the once and forever acquired data sets this combination of unbiased broad range precursor ion fragmentation and targeted data extraction alleviates most constraints of present proteomic method and should be equally applicable to the comprehensive analysis of other classes of analytes beyond proteomics

most proteomic studies use liquid chromatography coupled to tandem mass spectrometry to identify and quantify the peptide generated by the proteolysis of biological sample however with the current method it remains challenging to rapidly consistently reproducibly accurately and sensitively detect and quantify large fractions of proteomes across multiple samples here we present new strategy that systematically queries sample sets for the presence and quantity of essentially any protein of interest It consists of using the information available in fragment ion spectral libraries to mine the complete fragment ion maps generated using data independent acquisition method for this study the data were acquired on fast high resolution quadrupole quadrupole time of flight TOF instrument by repeatedly cycling through consecutive Da precursor isolation windows swaths this SWATH MS acquisition setup generates in single sample injection time resolved fragment ion spectra for all the analytes detectable within the precursor range and the user defined retention time window We show that suitable combinations of fragment ions extracted from these data sets are sufficiently specific to confidently identify query peptide over dynamic range of orders of magnitude even if the precursors of the queried peptide are not detectable in the survey scans We also show that queried peptide are quantified with consistency and accuracy comparable with that of selected reaction monitoring the gold standard proteomic quantification method moreover targeted data extraction enables ad libitum quantification refinement and dynamic extension of protein probing by iterative re mining of the once and forever acquired data sets this combination of unbiased broad range precursor ion fragmentation and targeted data extraction alleviates most constraints of present proteomic method and should be equally applicable to the comprehensive analysis of other classes of analytes beyond proteomics

selected reaction monitoring SRM is targeted mass spectrometry technique that is emerging in the field of proteomics as complement to untargeted shotgun method SRM is particularly useful when predetermined sets of protein such as those constituting cellular networks or sets of candidate biomarkers need to be measured across multiple samples in consistent reproducible and quantitatively precise manner here we describe how SRM is applied in proteomics review recent advances present selected applications and provide perspective on the future of this powerful technology

TRiC CCT is highly conserved and essential chaperonin that uses ATP cycling to facilitate folding of approximately of the eukaryotic proteome this MDa hetero oligomeric complex consists of two stacked rings of eight paralogous subunits each previously proposed TRiC models differ substantially in their subunit arrangements and ring register here we integrate chemical crosslinking mass spectrometry and combinatorial modeling to reveal the definitive subunit arrangement of TRiC In vivo disulfide mapping provided additional validation for the crosslinking derived arrangement as the definitive TRiC topology this subunit arrangement allowed the refinement of structural model using existing ray diffraction data the structure described here explains all available crosslink experiments provides rationale for previously unexplained structural features and reveals surprising asymmetry of charges within the chaperonin folding chamber

epithelial ovarian carcinoma has in general poor prognosis since the vast majority of tumors are genomically unstable and clinically highly aggressive this results in rapid progression of malignancy potential while still asymptomatic and thus in late diagnosis It is therefore of critical importance to develop method to diagnose epithelial ovarian carcinoma at its earliest developmental stage that is to differentiate between benign tissue and its early malignant transformed counterparts here we present shotgun quantitative proteomic screen of benign and malignant epithelial ovarian tumors using iTRAQ technology with LC MALDI TOF TOF and LC ESI QTOF MS MS pathway analysis of the shotgun data pointed to the PI3K akt signaling pathway as significant discriminatory pathway selected candidate protein from the shotgun screen were further confirmed in individual tissue samples of normal benign borderline or malignant origin using LC MRM analysis the MRM profile demonstrated significant differences between the four groups separating the normal tissue samples from all tumor groups as well as perfectly separating the benign and malignant tumors with ROC area of this work demonstrates the utility of using shotgun approach to filter out signature of few protein only that discriminates between the different sample groups

the objective of the international chromosome centric human proteome project HPP is to map and annotate all protein encoded by the gene on each human chromosome the HPP consortium was established to organize collaborative network among the research teams responsible for protein mapping of individual chromosomes and to identify compelling biological and genetic mechanisms influencing colocated gene and their protein products the HPP aims to foster the development of proteome analysis and integration of the findings from related molecular omics technology platforms through collaborations among universities industries and private research groups the HPP consortium leadership has elicited broad input for standard guidelines to manage these international efforts more efficiently by mobilizing existing resources and collaborative networks the HPP guidelines set out the collaborative consensus of the HPP teams introduce topics associated with experimental approaches data production quality control treatment and transparency of data governance of the consortium and collaborative benefits companion approach for the biology and disease driven HPP HPP component of the human proteome project is currently being organized building upon the human proteome organization organ based and biofluid based initiatives www hupo org research the common application of these guidelines in the participating laboratories is expected to facilitate the goal of comprehensive analysis of the human proteome

protein identifications instead of peptide spectrum matches constitute the biologically relevant result of shotgun proteomics studies how to appropriately infer and report protein identifications has triggered still ongoing debate this debate has so far suffered from the lack of appropriate performance measures that allow us to objectively assess protein inference approaches this study describes an intuitive generic and yet formal performance measure and demonstrates how it enables experimentalists to select an optimal protein inference strategy for given collection of fragment ion spectra We applied the performance measure to systematically explore the benefit of excluding possibly unreliable protein identifications such as single hit wonders therefore we defined family of protein inference engines by extending simple inference engine by thousands of pruning variants each excluding different specified set of possibly unreliable identifications We benchmarked these protein inference engines on several data sets representing different proteomes and mass spectrometry platforms optimally performing inference engines retained all high confidence spectral evidence without posterior exclusion of any type of protein identifications despite the diversity of studied data sets consistently supporting this rule other data sets might behave differently In order to ensure maximal reliable proteome coverage for data sets arising in other studies we advocate abstaining from rigid protein inference rules such as exclusion of single hit wonders and instead consider several protein inference approaches and assess these with respect to the presented performance measure in the specific application context

selected reaction monitoring SRM is targeted mass spectrometry technique that provides sensitive and accurate protein detection and quantification in complex biological mixtures statistical and computational tools are essential for the design and analysis of SRM experiments particularly in studies with large sample throughput currently most such tools focus on the selection of optimized transitions and on processing signals from SRM assay little attention is devoted to protein significance analysis which combines the quantitative measurements for protein across isotopic labels peptide charge states transitions samples and conditions and detects protein that change in abundance between conditions while controlling the false discovery rate We propose statistical modeling framework for protein significance analysis It is based on linear mixed effects models and is applicable to most experimental designs for both isotope label based and label free SRM workflows We illustrate the utility of the framework in two studies one with group comparison experimental design and the other with time course experimental design We further verify the accuracy of the framework in two controlled data sets one from the NCI CPTAC reproducibility investigation and the other from an in house spike in study the proposed framework is sensitive and specific produces accurate results in broad experimental circumstances and helps to optimally design future SRM experiments the statistical framework is implemented in an open source based software package SRmstats and can be used by researchers with limited statistics background as standalone tool or in integration with the existing computational pipelines

selected or multiple reaction monitoring is targeted mass spectrometry method MRM MS in which many peptide are simultaneously and consistently analyzed during single liquid chromatography mass spectrometry LC MRM MS measurement these capabilities make MRM MS an attractive method to monitor consistent set of protein over various experimental conditions To increase throughput for MRM MS it is advantageous to use scheduled method and unfractionated protein extracts here we established the practically measurable dynamic range of protein reliably detectable and quantifiable in an unfractionated protein extract from human cell line using LC MRM MS initially we analyzed MRM transition peak groups in terms of interfering signals and compared MRM transition peak groups to MS1 triggered MS2 spectra using dot product analysis finally using unfractionated protein extract from human cell lysate we quantified the upper boundary of copies per cell to be million copies per cell while copies per cell represents lower boundary using single min linear gradient LC MRM MS measurement on current standard commercial instrument

selected reaction monitoring SRM is targeted mass spectrometry technique that provides sensitive and accurate protein detection and quantification in complex biological mixtures statistical and computational tools are essential for the design and analysis of SRM experiments particularly in studies with large sample throughput currently most such tools focus on the selection of optimized transitions and on processing signals from SRM assay little attention is devoted to protein significance analysis which combines the quantitative measurements for protein across isotopic labels peptide charge states transitions samples and conditions and detects protein that change in abundance between conditions while controlling the false discovery rate We propose statistical modeling framework for protein significance analysis It is based on linear mixed effects models and is applicable to most experimental designs for both isotope label based and label free SRM workflows We illustrate the utility of the framework in two studies one with group comparison experimental design and the other with time course experimental design We further verify the accuracy of the framework in two controlled data sets one from the NCI CPTAC reproducibility investigation and the other from an in house spike in study the proposed framework is sensitive and specific produces accurate results in broad experimental circumstances and helps to optimally design future SRM experiments the statistical framework is implemented in an open source based software package SRmstats and can be used by researchers with limited statistics background as stand alone tool or in integration with the existing computational pipelines

protein identifications instead of peptide spectrum matches constitute the biologically relevant result of shotgun proteomics studies how to appropriately infer and report protein identifications has triggered still ongoing debate this debate has so far suffered from the lack of appropriate performance measures that allow us to objectively assess protein inference approaches this study describes an intuitive generic and yet formal performance measure and demonstrates how it enables experimentalists to select an optimal protein inference strategy for given collection of fragment ion spectra We applied the performance measure to systematically explore the benefit of excluding possibly unreliable protein identifications such as single hit wonders therefore we defined family of protein inference engines by extending simple inference engine by thousands of pruning variants each excluding different specified set of possibly unreliable identifications We benchmarked these protein inference engines on several data sets representing different proteomes and mass spectrometry platforms optimally performing inference engines retained all high confidence spectral evidence without posterior exclusion of any type of protein identifications despite the diversity of studied data sets consistently supporting this rule other data sets might behave differently In order to ensure maximal reliable proteome coverage for data sets arising in other studies we advocate abstaining from rigid protein inference rules such as exclusion of single hit wonders and instead consider several protein inference approaches and assess these with respect to the presented performance measure in the specific application context

targeted proteomics via selected reaction monitoring is powerful mass spectrometric technique affording higher dynamic range increased specificity and lower limits of detection than other shotgun mass spectrometry method when applied to proteome analyses however it involves selective measurement of predetermined analytes which requires more preparation in the form of selecting appropriate signatures for the protein and peptide that are to be targeted there is growing number of software programs and resources for selecting optimal transitions and the instrument settings used for the detection and quantification of the targeted peptide but the exchange of this information is hindered by lack of standard format We have developed new standardized format called traML for encoding transition lists and associated metadata In addition to introducing the traML format we demonstrate several implementations across the community and provide semantic validators extensive documentation and multiple example instances to demonstrate correctly written documents widespread use of traML will facilitate the exchange of transitions reduce time spent handling incompatible list formats increase the reusability of previously optimized transitions and thus accelerate the widespread adoption of targeted proteomics via selected reaction monitoring

targeted proteomics via selected reaction monitoring is powerful mass spectrometric technique affording higher dynamic range increased specificity and lower limits of detection than other shotgun mass spectrometry method when applied to proteome analyses however it involves selective measurement of predetermined analytes which requires more preparation in the form of selecting appropriate signatures for the protein and peptide that are to be targeted there is growing number of software programs and resources for selecting optimal transitions and the instrument settings used for the detection and quantification of the targeted peptide but the exchange of this information is hindered by lack of standard format We have developed new standardized format called traML for encoding transition lists and associated metadata In addition to introducing the traML format we demonstrate several implementations across the community and provide semantic validators extensive documentation and multiple example instances to demonstrate correctly written documents widespread use of traML will facilitate the exchange of transitions reduce time spent handling incompatible list formats increase the reusability of previously optimized transitions and thus accelerate the widespread adoption of targeted proteomics via selected reaction monitoring

public repositories for proteomics data have accelerated proteomics research by enabling more efficient cross analyses of datasets supporting the creation of protein and peptide compendia of experimental results supporting the development and testing of new software tools and facilitating the manuscript review process the repositories available to date have been designed to accommodate either shotgun experiments or generic proteomic data files here we describe new kind of proteomic data repository for the collection and representation of data from selected reaction monitoring SRM measurements the peptideatlas SRM experiment library PASSEL allows researchers to easily submit proteomic data sets generated by SRM the raw data are automatically processed in uniform manner and the results are stored in database where they may be downloaded or browsed via web interface that includes chromatogram viewer PASSElenables cross analysis of SRmdata supports optimization of SRmdata collection and facilitates the review process of SRmdata further PASSElwill help in the assessment of proteotypic peptide performance in wide array of samples containing the same peptide as well as across multiple experimental protocols

mammalian host response to pathogens is associated with fluctuations in high abundant protein in body fluids as well as in regulation of protein expressed in relatively low copy numbers like cytokines secreted from immune cell and endothelium hence efficient monitoring of protein associated with host response to pathogens remains challenging task In this paper we present targeted proteome analysis of panel of protein that are widely believed to be key players and indicators of bovine host response to mastitis pathogens stable isotope labeled variants of two concordant proteotypic peptide from each of these protein were obtained through the qconCAT method We present the quantotypic properties of these proteotypic peptide and discuss their application to research in host pathogen interactions our results clearly demonstrate robust monitoring of targeted host response protein twelve of these were readily quantified in simple extraction of mammary gland tissues while the expression levels of the remaining protein were too low for direct and stable quantification hence their accurate quantification requires further fractionation of mammary gland tissues

targeted proteomics allows researchers to study protein of interest without being drowned in data from other less interesting protein or from redundant or uninformative peptide while the technique is mostly used for smaller focused studies there are several reasons to conduct larger targeted experiments automated highly robust software becomes more important in such experiments In addition larger experiments are carried out over longer periods of time requiring strategies to handle the sometimes large shift in retention time often observed We present complete proof of principle software stack that automates most aspects of selected reaction monitoring workflows targeted proteomics technology the software allows experiments to be easily designed and carried out the steps automated are the generation of assay generation of mass spectrometry driver files and method files and the import and analysis of the data all data are normalized to common retention time scale the data are then scored using novel score model and the error is subsequently estimated We also show that selected reaction monitoring can be used for label free quantification all data generated are stored in relational database and the growing resource further facilitates the design of new experiments We apply the technology to large scale experiment studying how streptococcus pyogenes remodels its proteome under stimulation of human plasma

cell surface glycoproteins provide key interface of cell to their environment and therapeutic entry points for drug and biomarker discovery their comprehensive description denotes therefore formidable challenge the cell of the pancreas play crucial role in blood glucose homeostasis and disruption of their function contributes to diabetes By combining cell surface and whole cell capturing technologies with high throughput quantitative proteomic analysis we report on the identification of total of unique glycoproteins from mouse MIN6 cell and human islets three hundred forty nine of these protein encompass potential surface glycoproteins and include orphan protein coupled receptors novel proteases receptor protein kinases and phosphatases interestingly stimulation of MIN6 cell with glucose and the hormone GLP1 known stimulators of insulin secretion causes significant changes in surface glycoproteome expression taken together this cell glycoproteome resource provides comprehensive view on the composition of cell surface protein and expands the scope of signaling system potentially involved in mediating responses of cell to various forms of patho physiologic stress and the extent of dynamic remodeling of surface glycoprotein expression associated with metabolic and hormonal stimulation moreover it provides foundation for the development of diabetes medicines that target or are derived from the cell surface glycoproteome

for many research questions in modern molecular and system biology information about absolute protein quantities is imperative this information includes for example kinetic modeling of process protein turnover determinations stoichiometric investigations of protein complexes or quantitative comparisons of different protein within one sample or across samples To date the vast majority of proteomic studies are limited to providing relative quantitative comparisons of protein levels between limited numbers of samples here we describe and demonstrate the utility of targeting MS technique for the estimation of absolute protein abundance in unlabeled and nonfractionated cell lysates the method is based on selected reaction monitoring SRM mass spectrometry and the best flyer hypothesis which assumes that the specific MS signal intensity of the most intense tryptic peptide per protein is approximately constant throughout whole proteome SRM targeted best flyer peptide were selected for each protein from the peptide precursor ion signal intensities from directed MS data the most intense transitions per peptide were selected from full MS MS scans of crude synthetic analogs We used monte carlo cross validation to systematically investigate the accuracy of the technique as function of the number of measured best flyer peptide and the number of SRM transitions per peptide We found that linear model based on the two most intense transitions of the three best flying peptide per protein toppep3 toptra2 generated optimal results with cross correlated mean fold error of and squared pearson coefficient R2 of applying the optimized model to lysates of the microbe leptospira interrogans we detected significant protein abundance changes of target protein upon antibiotic treatment which correlate well with literature values the described method is generally applicable and exploits the inherent performance advantages of SRM such as high sensitivity selectivity reproducibility and dynamic range and estimates absolute protein concentrations of selected protein at minimized costs

protein assemblies are critical for cellular function and understanding their physical organization is the key aim of structural biology however applying conventional structural biology approaches is challenging for transient dynamic or polydisperse assemblies there is therefore growing demand for hybrid technologies that are able to complement classical structural biology method and thereby broaden our arsenal for the study of these important complexes exciting new developments in the field of mass spectrometry and proteomics have added new dimension to the study of protein protein interactions and protein complex architecture In this review we focus on how complementary mass spectrometry based techniques can greatly facilitate structural understanding of protein assemblies

for many research questions in modern molecular and system biology information about absolute protein quantities is imperative this information includes for example kinetic modeling of process protein turnover determinations stoichiometric investigations of protein complexes or quantitative comparisons of different protein within one sample or across samples To date the vast majority of proteomic studies are limited to providing relative quantitative comparisons of protein levels between limited numbers of samples here we describe and demonstrate the utility of targeting MS technique for the estimation of absolute protein abundance in unlabeled and nonfractionated cell lysates the method is based on selected reaction monitoring SRM mass spectrometry and the best flyer hypothesis which assumes that the specific MS signal intensity of the most intense tryptic peptide per protein is approximately constant throughout whole proteome SRM targeted best flyer peptide were selected for each protein from the peptide precursor ion signal intensities from directed MS data the most intense transitions per peptide were selected from full MS MS scans of crude synthetic analogs We used monte carlo cross validation to systematically investigate the accuracy of the technique as function of the number of measured best flyer peptide and the number of SRM transitions per peptide We found that linear model based on the two most intense transitions of the three best flying peptide per protein toppep3 toptra2 generated optimal results with cross correlated mean fold error of and squared pearson coefficient of applying the optimized model to lysates of the microbe leptospira interrogans we detected significant protein abundance changes of target protein upon antibiotic treatment which correlate well with literature values the described method is generally applicable and exploits the inherent performance advantages of SRM such as high sensitivity selectivity reproducibility and dynamic range and estimates absolute protein concentrations of selected protein at minimized costs

chemical cross linking in combination with mass spectrometric analysis offers the potential to obtain low resolution structural information from protein and protein complexes identification of peptide connected by cross link provides direct evidence for the physical interaction of amino-acid side chains information that can be used for computational modeling purposes despite impressive advances that were made in recent years the number of experimentally observed cross links still falls below the number of possible contacts of cross linkable side chains within the span of the cross linker here we propose two complementary experimental strategies to expand cross linking data sets first enrichment of cross linked peptide by size exclusion chromatography selects cross linked peptide based on their higher molecular mass thereby depleting the majority of unmodified peptide present in proteolytic digests of cross linked samples second we demonstrate that the use of proteases in addition to trypsin such as asp can additionally boost the number of observable cross linking sites the benefits of both SEC enrichment and multiprotease digests are demonstrated on set of model protein and the improved workflow is applied to the characterization of the 20S proteasome from rabbit and schizosaccharomyces pombe

the term proteomics encompasses the large scale detection and analysis of protein and their post translational modifications driven by major improvements in mass spectrometric instrumentation methodology and data analysis the proteomics field has burgeoned in recent years It now provides range of sensitive and quantitative approaches for measuring protein structures and dynamics that promise to revolutionize our understanding of cell biology and molecular mechanisms in both human cell and model organisms the proteomics specification in time and space PROSPECTS network is unique EU funded project that brings together leading european research groups spanning from instrumentation to biomedicine in collaborative five year initiative to develop new method and applications for the functional analysis of cellular protein this special issue of molecular and cellular proteomics presents research papers reporting major recent progress by the PROSPECTS groups including improvements to the resolution and sensitivity of the orbitrap family of mass spectrometers systematic detection of protein using highly characterized antibody collections and new method for absolute as well as relative quantification of protein levels manuscripts in this issue exemplify approaches for performing quantitative measurements of cell proteomes and for studying their dynamic responses to perturbation both during normal cellular responses and in disease mechanisms here we present perspective on how the proteomics field is moving beyond simply identifying protein with high sensitivity toward providing powerful and versatile set of assay system for characterizing proteome dynamics and thereby creating new third generation proteomics strategy that offers an indispensible tool for cell biology and molecular medicine

protein assemblies are critical for cellular function and understanding their physical organization is the key aim of structural biology however applying conventional structural biology approaches is challenging for transient dynamic or polydisperse assemblies there is therefore growing demand for hybrid technologies that are able to complement classical structural biology method and thereby broaden our arsenal for the study of these important complexes exciting new developments in the field of mass spectrometry and proteomics have added new dimension to the study of protein protein interactions and protein complex architecture In this review we focus on how complementary mass spectrometry based techniques can greatly facilitate structural understanding of protein assemblies

chemical cross linking in combination with mass spectrometric analysis offers the potential to obtain low resolution structural information from protein and protein complexes identification of peptide connected by cross link provides direct evidence for the physical interaction of amino-acid side chains information that can be used for computational modeling purposes despite impressive advances that were made in recent years the number of experimentally observed cross links still falls below the number of possible contacts of cross linkable side chains within the span of the cross linker here we propose two complementary experimental strategies to expand cross linking data sets first enrichment of cross linked peptide by size exclusion chromatography selects cross linked peptide based on their higher molecular mass thereby depleting the majority of unmodified peptide present in proteolytic digests of cross linked samples second we demonstrate that the use of proteases in addition to trypsin such as asp can additionally boost the number of observable cross linking sites the benefits of both SEC enrichment and multiprotease digests are demonstrated on set of model protein and the improved workflow is applied to the characterization of the 20S proteasome from rabbit and schizosaccharomyces pombe

the community working on model organisms is growing steadily and the number of model organisms for which proteome data are being generated is continuously increasing To standardize efforts and to make optimal use of proteomics data acquired from model organisms new human proteome organisation HUPO initiative on model organism proteomes iMOP was approved at the HUPO ninth annual world congress in sydney iMOP will seek to stimulate scientific exchange and disseminate HUPO best practices the needs of model organism researchers for central databases will be better represented catalyzing the integration of proteomics and organism specific databases full details of iMOP activities members tools and resources can be found at our website and new members are invited to join us

the 26S proteasome is at the executive end of the ubiquitinproteasome pathway for the controlled degradation of intracellular protein while the structure of its 20S core particle CP has been determined by ray crystallography the structure of the 19S regulatory particle RP which recruits substrates unfolds them and translocates them to the CP for degradation has remained elusive here we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy ray crystallography residue specific chemical cross linking and several proteomics techniques the lid of the RP consisting of rpn3 is organized in modular fashion rpn3 form horseshoe shaped heterohexamer which connects to the CP and roofs the AAA ATpase module positioning the rpn8 rpn11 heterodimer close to its mouth rpn2 is rigid supporting the lid while rpn1 is conformationally variable positioned at the periphery of the ATpase ring the ubiquitin receptors rpn10 and rpn13 are located in the distal part of the RP indicating that they were recruited to the complex late in its evolution the modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates

streptococcus pyogenes is major bacterial pathogen and potent inducer of inflammation causing plasma leakage at the site of infection combination of label free quantitative mass spectrometry based proteomics strategies were used to measure how the intracellular proteome homeostasis of pyogenes is influenced by the presence of human plasma identifying and quantifying protein In plasma the bacterium modifies its production of protein and the most pronounced change was the complete down regulation of protein required for fatty acid biosynthesis fatty acids are transported by albumin HSA in plasma pyogenes expresses HSA binding surface protein and HSA carrying fatty acids reduced the amount of fatty acid biosynthesis protein to the same extent as plasma the results clarify the function of HSA binding protein in pyogenes and underline the power of the quantitative mass spectrometry strategy used here to investigate bacterial adaptation to given environment

In high throughput mass spectrometry proteomics peptide and protein are not simply identified as present or not present in sample rather the identifications are associated with differing levels of confidence the false discovery rate FDR has emerged as an accepted means for measuring the confidence associated with identifications We have developed the systematic protein investigative research environment SPIRE for the purpose of integrating the best available proteomics method two successful approaches to estimating the FDR for MS protein identifications are the MAYU and our current SPIRE method We present here method to combine these two approaches to estimating the FDR for MS protein identifications into an integrated protein model IPM We illustrate the high quality performance of this IPM approach through testing on two large publicly available proteomics datasets MAYU and SPIRE show remarkable consistency in identifying protein in these datasets still IPM results in more robust FDR estimation approach and additional identifications particularly among low abundance protein IPM is now implemented as part of the SPIRE system

background modern data generation techniques used in distributed system biology research projects often create datasets of enormous size and diversity We argue that in order to overcome the challenge of managing those large quantitative datasets and maximise the biological information extracted from them sound information system is required ease of integration with data analysis pipelines and other computational tools is key requirement for it results We have developed openBIS an open source software framework for constructing user friendly scalable and powerful information system for data and metadata acquired in biological experiments openBIS enables users to collect integrate share publish data and to connect to data processing pipelines this framework can be extended and has been customized for different data types acquired by range of technologies conclusions openBIS is currently being used by several systemsX ch and EU projects applying mass spectrometric measurements of metabolites and protein high content screening or next generation sequencing technologies the attributes that make it interesting to large research community involved in system biology projects include versatility simplicity in deployment scalability to very large data flexibility to handle any biological data type and extensibility to the needs of any research domain

background metastatic castration resistant prostate cancer mCRPC is associated with poor outcome prognostic information is useful and aids treatment decisions however current nomograms based on clinical parameters alone have weak prognostic accuracy therefore the identification of new prognostic serum biomarkers could be useful objectives To assess if quantitative analysis of the phosphatase and tensin homolog pten conditional knockout mouse proteome reveals significant prognostic biomarkers in mCRPC and to compare the accuracy of these biomarkers with known prognostic factors design setting and participants fifty seven patients with mCRPC were evaluated retrospectively prognostic factors used in clinical nomograms were assessed from the records new candidate biomarkers in patients sera were derived using cancer genetics guided model we recently described screening the murine pten dependent glycoproteome measurements quantification in patients sera was performed by either mass spectrometry based targeted proteomics or enzyme linked immunosorbent assay prognostic biomarkers for survival were identified based on kaplan meier models In second step random forest analysis was performed to identify prognostic signature combined from the pooled data of known predictors and newly identified biomarkers results and limitations with univariate analysis new significant prognostic factors for survival in the sera of mCRPC patients were found with bonferroni corrected level of significance random forest analysis revealed five factor predictor thrombospondin reactive protein poliovirus receptor related ephrin A5 and membrane metallo endopeptidase with an accuracy of and for and mo survival respectively this means that in our dataset the error was reduced by compared to using the halabi et al nomogram the retrospective nature of the work and absence of validating dataset is the major limitation of this work conclusions analysis of the serum proteome in mCRPC patients based on our pten conditional knockout model combined with known prognostic factors potentially improves accuracy of prognostic nomograms these newly identified markers have to be validated in prospective studies

peptideatlas is web accessible database of LC MS MS shotgun proteomics results from hundreds of experiments conducted in diverse laboratories with all data processed via uniform analysis pipeline total of experiments on human plasma and serum are included using the peptideatlas web interface users can browse and search the human plasma peptideatlas for identified peptide and identified protein view spectra and select proteotypic peptide users can easily view supporting information such as chromosomal mapping estimated abundances and sequence alignments herein the reader is instructed in the use of the human plasma peptideatlas through an illustrated exploration of cytokine receptors in plasma

the combination of tandem mass spectrometry and sequence database searching is the method of choice for the identification of peptide and the mapping of proteomes over the last several years the volume of data generated in proteomic studies has increased dramatically which challenges the computational approaches previously developed for these data furthermore multitude of search engines have been developed that identify different overlapping subsets of the sample peptide from particular set of tandem mass spectrometry spectra We present iprophet the new addition to the widely used open source suite of proteomic data analysis tools trans proteomics pipeline applied in tandem with peptideprophet it provides more accurate representation of the multilevel nature of shotgun proteomic data iprophet combines the evidence from multiple identifications of the same peptide sequence across different spectra experiments precursor ion charge states and modified states It also allows accurate and effective integration of the results from multiple database search engines applied to the same data the use of iprophet in the trans proteomics pipeline increases the number of correctly identified peptide at constant false discovery rate as compared with both peptideprophet and another state of the art tool percolator As the main outcome iprophet permits the calculation of accurate posterior probabilities and false discovery rate estimates at the level of sequence identical peptide identifications which in turn leads to more accurate probability estimates at the protein level fully integrated with the trans proteomics pipeline it supports all commonly used MS instruments search engines and computer platforms the performance of iprophet is demonstrated on two publicly available data sets data from human whole cell lysate proteome profiling experiment representative of typical proteomic data sets and from set of streptococcus pyogenes experiments more representative of organism specific composite data sets

targeted mass spectrometry using selected reaction monitoring SRM has emerged as the method of choice for the validation in blood serum plasma or other clinically relevant specimens of biomarker candidates arising from comparative proteomics or other discovery strategies here we describe method in which glycosites are selectively enriched from biological specimens by solid phase capture and PNgase release and then analyzed by SRM focusing the highly sensitive targeted mass spectrometry method on subproteome enriched for secreted and shed protein reproducibly identifies and quantifies such protein in serum and plasma at the low nanogram per milliliter ng mL concentration range this protocol is intended to give an introduction to SRM based targeted mass spectrometry with special focus on the validation of biomarker candidates

the combination of tandem mass spectrometry and sequence database searching is the method of choice for the identification of peptide and the mapping of proteomes over the last several years the volume of data generated in proteomic studies has increased dramatically which challenges the computational approaches previously developed for these data furthermore multitude of search engines have been developed that identify different overlapping subsets of the sample peptide from particular set of tandem mass spectrometry spectra We present iprophet the new addition to the widely used open source suite of proteomic data analysis tools trans proteomics pipeline applied in tandem with peptideprophet it provides more accurate representation of the multilevel nature of shotgun proteomic data iprophet combines the evidence from multiple identifications of the same peptide sequence across different spectra experiments precursor ion charge states and modified states It also allows accurate and effective integration of the results from multiple database search engines applied to the same data the use of iprophet in the trans proteomics pipeline increases the number of correctly identified peptide at constant false discovery rate as compared with both peptideprophet and another state of the art tool percolator As the main outcome iprophet permits the calculation of accurate posterior probabilities and false discovery rate estimates at the level of sequence identical peptide identifications which in turn leads to more accurate probability estimates at the protein level fully integrated with the trans proteomics pipeline it supports all commonly used MS instruments search engines and computer platforms the performance of iprophet is demonstrated on two publicly available data sets data from human whole cell lysate proteome profiling experiment representative of typical proteomic data sets and from set of streptococcus pyogenes experiments more representative of organismspecific composite data sets

nutrient sensing and coordination of metabolic pathways are crucial functions for all living cell but details of the coordination under different environmental conditions remain elusive We therefore undertook system biology approach to investigate the interactions between the snf1 and the target of rapamycin complex TORC1 in saccharomyces cerevisiae We show that snf1 regulates much broader range of biological process compared with TORC1 under both glucose and ammonium limited conditions We also find that snf1 has role in upregulating the NADP + dependent glutamate dehydrogenase encoded by GDH3 under derepressing condition and therefore may also have role in ammonium assimilation and amino-acid biosynthesis which can be considered as convergence of snf1 and TORC1 pathways In addition to the accepted role of snf1 in regulating fatty acid FA metabolism we show that TORC1 also regulates FA metabolism likely through modulating the peroxisome and oxidation finally we conclude that direct interactions between snf1 and TORC1 pathways are unlikely under nutrient limited conditions and propose that TORC1 is repressed in manner that is independent of snf1

genetic analysis in drosophila melanogaster has been widely used to identify system of gene that control cell growth in response to insulin and nutrients many of these gene encode components of the insulin receptor target of rapamycin InR TOR pathway however the biochemical context of this regulatory system is still poorly characterized in drosophila here we present the first quantitative study that systematically characterizes the modularity and hormone sensitivity of the interaction proteome underlying growth control by the dInR TOR pathway applying quantitative affinity purification and mass spectrometry we identified high confidence protein interactions among network components In all of the detected interactions were regulated by insulin affecting membrane proximal as well as intracellular signaling complexes systematic functional analysis linked subset of network components to the control of dTORC1 and dTORC2 activity furthermore our data suggest the presence of three distinct dTOR kinase complexes including the evolutionary conserved dTTT complex drosophila TOR TELO2 TTI1 subsequent genetic studies in flies suggest role for dTTT in controlling cell growth via dTORC1 and dTORC2 dependent mechanism

the generation of mathematical models of biological process the simulation of these process under different conditions and the comparison and integration of multiple data sets are explicit goals of system biology that require the knowledge of the absolute quantity of the system components To date systematic estimates of cellular protein concentrations have been exceptionally scarce here we provide quantitative description of the proteome of commonly used human cell line in two functional states interphase and mitosis We show that these human cultured cell express at least protein and that the quantified protein span concentration range of seven orders of magnitude up to copies per cell We discuss how protein abundance is linked to function and evolution

prediction of the responses to neoadjuvant chemotherapy NACT can improve the treatment of patients with advanced breast cancer gene and protein predictive of chemoresistance have been extensively studied in breast cancer tissues however noninvasive serum biomarkers capable of such prediction have been rarely exploited here we performed profiling of glycosylated protein in serum from fifteen advanced breast cancer patients ten patients sensitive to and five patients resistant to NACT to discover serum biomarkers of chemoresistance using label free liquid chromatography tandem MS method By performing series of statistical analyses of the proteomic data we selected thirteen biomarker candidates and tested their differential serum levels by western blotting in independent samples eight patients sensitive to and five patients resistant to NACT among the candidates we then selected the final set of six potential serum biomarkers AHSG APOB C3 C9 CP and ORM1 whose differential expression was confirmed in the independent samples finally we demonstrated that multivariate classification model using the six protein could predict responses to NACT and further predict relapse free survival of patients In summary global glycoproteome profile in serum revealed protein pattern predictive of the responses to NACT which can be further validated in large clinical studies

prediction of the responses to neoadjuvant chemotherapy NACT can improve the treatment of patients with advanced breast cancer gene and protein predictive of chemoresistance have been extensively studied in breast cancer tissues however noninvasive serum biomarkers capable of such prediction have been rarely exploited here we performed profiling of glycosylated protein in serum from fifteen advanced breast cancer patients ten patients sensitive to and five patients resistant to NACT to discover serum biomarkers of chemoresistance using label free liquid chromatography tandem MS method By performing series of statistical analyses of the proteomic data we selected thirteen biomarker candidates and tested their differential serum levels by western blotting in independent samples eight patients sensitive to and five patients resistant to NACT among the candidates we then selected the final set of six potential serum biomarkers AHSG APOB C3 C9 CP and ORM1 whose differential expression was confirmed in the independent samples finally we demonstrated that multivariate classification model using the six protein could predict responses to NACT and further predict relapse free survival of patients In summary global glycoproteome profile in serum revealed protein pattern predictive of the responses to NACT which can be further validated in large clinical studies

cell growth is regulated during RNA polymerase pol transcription initiation by the conserved factor rrn3 TIF IA in yeast humans here we provide structure function analysis of rrn3 based on combination of structural biology with in vivo and in vitro functional assay the rrn3 crystal structure reveals unique HEAT repeat fold and surface serine patch phosphorylation of this patch represses human pol transcription and phospho mimetic patch mutation prevents rrn3 binding to pol in vitro and reduces cell growth and pol gene occupancy in vivo cross linking indicates that rrn3 binds pol between its subcomplexes AC40 and A14 which faces the serine patch the corresponding region of pol II binds the mediator head that cooperates with transcription factor TF IIB consistent with this the rrn3 binding factor rrn7 is predicted to be TFIIB homolog this reveals the molecular basis of rrn3 regulated pol initiation and cell growth and indicates general architecture of eukaryotic transcription initiation complexes

decreased cell mass and function are hallmarks of type diabetes here we identified through siRNA screen beta site amyloid precursor protein cleaving enzyme bace2 as the sheddase of the proproliferative plasma membrane protein tmem27 in murine and human cell mice with functionally inactive bace2 and insulin resistant mice treated with newly identified bace2 inhibitor both display augmented cell mass and improved control of glucose homeostasis due to increased insulin levels these results implicate bace2 in the control of cell maintenance and provide rational strategy to inhibit this protease for the expansion of functional pancreatic cell mass

lung cancer is the leading cause of all cancer related deaths with worldwide mortality of million each year the year survival rate ranges from in early stages to dismal in advanced disease prognosis is currently mostly determined based on the extension of disease at diagnosis thereby it has become evident that predicted and real outcomes can vary significantly even for patients with the same stage of disease novel biomarkers with reliable predictive significance are therefore clearly needed In this study we implemented an activity based solely mass spectrometry dependent biomarker discovery platform We investigated the role of serine hydrolase activities as potential biomarkers for human lung adenocarcinoma the most common lung cancer subtype forty pairs of fresh frozen malignant and matching non neoplastic lung tissues were analyzed and enzymatic activities linked to clinical follow up data We found that the activities of abhydrolase domain containing protein and esterase predict the development of distant metastases and the presence of aggressive lung adenocarcinomas respectively in statistically significant model We conclude that serine hydrolase activities bear predictive potential for human lung adenocarcinoma and that activity based proteomics represents powerful methodology in the search for novel disease biomarkers

human blood plasma can be obtained relatively noninvasively and contains protein from most if not all tissues of the body therefore an extensive quantitative catalog of plasma protein is an important starting point for the discovery of disease biomarkers In we showed that different proteomics measurements using different sample preparation and analysis techniques identify significantly different sets of protein and that comprehensive plasma proteome can be compiled only by combining data from many different experiments applying advanced computational method developed for the analysis and integration of very large and diverse data sets generated by tandem MS measurements of tryptic peptide we have now compiled high confidence human plasma proteome reference set with well over twice the identified protein of previous high confidence sets It includes hierarchy of protein identifications at different levels of redundancy following clearly defined scheme which we propose as standard that can be applied to any proteomics data set to facilitate cross proteome analyses further to aid in development of blood based diagnostics using techniques such as selected reaction monitoring we provide rough estimate of protein concentrations using spectral counting We identified distinct peptide from which we inferred highly nonredundant set of protein sequence at false discovery rate of We have made this resource available via peptideatlas large multiorganism publicly accessible compendium of peptide identified in tandem MS experiments conducted by laboratories around the world

receptor tyrosine kinases play essential roles in tissue development and homeostasis and aberrant signaling by these molecules is the basis of many diseases understanding the activation mechanism of these receptors is thus of high clinical relevance We investigated vascular endothelial growth factors VEGFs and their receptors VEGFRs which regulate blood and lymph vessel formation We analyzed the structural changes in the extracellular receptor domain that were induced by ligand binding and that represent the initial step in transmembrane signaling culminating in the activation of the intracellular receptor kinase domain high resolution structural information for the ligand binding domain became available recently but the flexibility of the extracellular domain and inhomogeneous glycosylation of VEGFRs have prevented the production of highly diffracting crystals of the entire extracellular domain so far therefore we chose to further investigate VEGFR structure by small angle ray scattering in solution SAXS SAXS data were combined with independent distance restraint determination obtained by mass spectrometric analysis of chemically cross linked ligand receptor complexes with these data we constructed structural model of the entire extracellular receptor domain in the unbound form and in complex with VEGF

human blood plasma can be obtained relatively noninvasively and contains protein from most if not all tissues of the body therefore an extensive quantitative catalog of plasma protein is an important starting point for the discovery of disease biomarkers In we showed that different proteomics measurements using different sample preparation and analysis techniques identify significantly different sets of protein and that comprehensive plasma proteome can be compiled only by combining data from many different experiments applying advanced computational method developed for the analysis and integration of very large and diverse data sets generated by tandem MS measurements of tryptic peptide we have now compiled high confidence human plasma proteome reference set with well over twice the identified protein of previous high confidence sets It includes hierarchy of protein identifications at different levels of redundancy following clearly defined scheme which we propose as standard that can be applied to any proteomics data set to facilitate cross proteome analyses further to aid in development of blood based diagnostics using techniques such as selected reaction monitoring we provide rough estimate of protein concentrations using spectral counting We identified distinct peptide from which we inferred highly nonredundant set of protein sequence at false discovery rate of We have made this resource available via peptideatlas large multiorganism publicly accessible compendium of peptide identified in tandem MS experiments conductedby laboratories around the world

the AAA ATpase VCP also known as p97 cooperates with distinct cofactors to process ubiquitylated protein in different cellular pathways VCP missense mutations cause systemic degenerative disease in humans but the molecular pathogenesis is unclear We used an unbiased mass spectrometry approach and identified VCP complex with the UBXD1 cofactor which binds to the plasma membrane protein caveolin CAV1 and whose formation is specifically disrupted by disease associated mutations We show that VCPg UBXD1 targets mono ubiquitylated CAV1 in SDS resistant high molecular weight complexes on endosomes which are en route to degradation in endolysosomes expression of VCP mutant protein chemical inhibition of VCP or siRNA mediated depletion of UBXD1 leads to block of CAV1 transport at the limiting membrane of enlarged endosomes in cultured cell In patient muscle muscle specific caveolin accumulates in sarcoplasmic pools and specifically delocalizes from the sarcolemma these results extend the cellular functions of VCP to mediating sorting of ubiquitylated cargo in the endocytic pathway and indicate that impaired trafficking of caveolin may contribute to pathogenesis in individuals with VCP mutations

We hereby provide two year update on the HUPO human plasma proteome project HPPP informed by advances presented at the HPPP sessions at the HUPO world congresses in toronto in september and in sydney in september copyright

the rapidly increasing ability to sequence complete genomes of different microbial species and strains provides us with information regarding their genetic variability genetic variability is mechanism for human pathogens to adapt to and avoid the immune system and to also develop resistance to antibiotics however the assessment of the contributions of individual genetic differences to resistance or other phenotypes is not priori apparent from the genomic variability quantitative proteomics can provide accurate molecular phenotypes of microbes that are difficult to determine using alternative technologies over the recent few years we and others have developed range of proteomic technologies for the quantitative analysis of microbial proteomes here we describe the most commonly used techniques and discuss their strengths and weaknesses and illustrate their respective performance for the identification of virulence factors in streptococcus pyogenes

end binding protein EBs comprise conserved family of microtubule plus end tracking protein the concerted action of calponin homology CH linker and terminal domains of EBs is important for their autonomous microtubule tip tracking regulation of microtubule dynamics and recruitment of numerous partners to microtubule ends here we report the detailed structural and biochemical analysis of mammalian EBs small angle ray scattering electron microscopy and chemical cross linking in combination with mass spectrometry indicate that EBs are elongated molecules with two interacting CH domains an arrangement reminiscent of that seen in other microtubule and actin binding protein removal of the negatively charged terminal tail did not affect the overall conformation of EBs however it increased the dwell times of EBs on the microtubule lattice in microtubule tip tracking reconstitution experiments An even more stable association with the microtubule lattice was observed when the entire negatively charged terminal domain of EBs was replaced by neutral coiled coil motif In contrast the interaction of EBs with growing microtubule tips was not significantly affected by these terminal domain mutations our data indicate that long range electrostatic repulsive interactions between the terminus and the microtubule lattice drive the specificity of EBs for growing microtubule ends

motivation chemical cross linking of protein or protein complexes and the mass spectrometry based localization of the cross linked amino-acid in peptide sequence is powerful method for generating distance restraints on the substrate topology results here we introduce the algorithm xwalk for predicting and validating these cross links on existing protein structures xwalk calculates and displays non linear distances between chemically cross linked amino-acid on protein surfaces while mimicking the flexibility and non linearity of cross linker molecules It returns solvent accessible surface distance which corresponds to the length of the shortest path between two amino-acid where the path leads through solvent occupied space without penetrating the protein surface

spectral library searching is an emerging approach in peptide identifications from tandem mass spectra critical step in proteomic data analysis In spectral library searching spectral library is first meticulously compiled from large collection of previously observed peptide MS MS spectra that are conclusively assigned to their corresponding amino-acid sequence An unknown spectrum is then identified by comparing it to all the candidates in the spectral library for the most similar match this review discusses the basic principles of spectral library building and searching describes its advantages and limitations and provides primer for researchers interested in adopting this new approach in their data analysis It will also discuss the future outlook on the evolution and utility of spectral libraries in the field of proteomics

over the past decade liquid chromatography coupled with tandem mass spectrometry LC MS MS has evolved into the main proteome discovery technology Up to several thousand protein can now be reliably identified from sample and the relative abundance of the identified protein can be determined across samples however the remeasurement of substantially similar proteomes for example those generated by perturbation experiments in system biology at high reproducibility and throughput remains challenging here we apply directed MS strategy to detect and quantify sets of pre determined peptide in tryptic digests of cell of the human pathogen leptospira interrogans at different states We show that in single LC MS MS experiment around peptide covering interrogans protein can be consistently detected and their absolute expression levels estimated revealing new insights about the proteome changes involved in pathogenic progression and antibiotic defense of interrogans this is the first study that describes the absolute quantitative behavior of any proteome over multiple states and represents the most comprehensive proteome abundance pattern comparison for any organism to date

biological function and cellular responses to environmental perturbations are regulated by complex interplay of DNA RNA protein and metabolites inside cell To understand these central process in living system at the molecular level we integrated experimentally determined abundance data for mRNA protein as well as individual protein half lives from the genome reduced bacterium mycoplasma pneumoniae We provide fine grained quantitative analysis of basic intracellular process under various external conditions proteome composition changes in response to cellular perturbations reveal specific stress response strategies the regulation of gene expression is largely decoupled from protein dynamics and translation efficiency has higher regulatory impact on protein abundance than protein turnover stochastic simulations using in vivo data show how low translation efficiency and long protein half lives effectively reduce biological noise in gene expression protein abundances are regulated in functional units such as complexes or pathways and reflect cellular lifestyles our study provides detailed integrative analysis of average cellular protein abundances and the dynamic interplay of mRNA and protein the central biomolecules of cell

after the successful completion of the human genome project the human proteome organization has recently officially launched global human proteome project HPP which is designed to map the entire human protein set given the lack of protein level evidence for about of the estimated protein coding gene systematic global effort will be necessary to achieve this goal with respect to protein abundance distribution subcellular localization interaction with other biomolecules and functions at specific time points As general experimental strategy HPP research groups will use the three working pillars for HPP mass spectrometry antibody capture and bioinformatics tools and knowledge bases the HPP participants will take advantage of the output and cross analyses from the ongoing human proteome organization initiatives and chromosome centric protein mapping strategy termed HPP with which many national teams are currently engaged In addition numerous biologically driven and disease oriented projects will be stimulated and facilitated by the HPP timely planning with proper governance of HPP will deliver protein parts list reagents and tools for protein studies and analyses and stronger basis for personalized medicine the human proteome organization urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in HPP consortium of funders and investigators

the DNA of all organisms is constantly subjected to damaging agents both exogenous and endogenous one extremely harmful lesion is the double strand break DSB which activates massive signaling network the DNA damage response DDR the chief activator of the DSB response is the ATM protein kinase which phosphorylates numerous key players in its various branches recent phosphoproteomic screens have extended the scope of damage induced phosphorylations beyond the direct ATM substrates We review the evidence for the involvement of numerous other protein kinases in the DDR obtained from documentation of specific pathways as well as high throughput screens the emerging picture of the protein phosphorylation landscape in the DDR broadens the current view on the role of this protein modification in the maintenance of genomic stability extensive cross talk between many of these protein kinases forms an interlaced signaling network that spans numerous cellular process versatile protein kinases in this network affect pathways that are different from those they have been identified with to date the DDR appears to be one of the most extensive signaling responses to cellular stimuli

selected reaction monitoring SRM is targeted mass spectrometric method that is increasingly used in proteomics for the detection and quantification of sets of preselected protein at high sensitivity reproducibility and accuracy currently data from SRM measurements are mostly evaluated subjectively by manual inspection on the basis of ad hoc criteria precluding the consistent analysis of different data sets and an objective assessment of their error rates here we present mprophet fully automated system that computes accurate error rates for the identification of targeted peptide in SRM data sets and maximizes specificity and sensitivity by combining relevant features in the data into statistical model

background since its inception proteomics has essentially operated in discovery mode with the goal of identifying and quantifying the maximal number of protein in sample increasingly proteomic measurements are also supporting hypothesis driven studies in which predetermined set of protein is consistently detected and quantified in multiple samples selected reaction monitoring SRM is targeted mass spectrometric technique that supports the detection and quantification of specific protein in complex samples at high sensitivity and reproducibility here we describe ATAQS an integrated software platform that supports all stages of targeted SRM based proteomics experiments including target selection transition optimization and post acquisition data analysis this software will significantly facilitate the use of targeted proteomic techniques and contribute to the generation of highly sensitive reproducible and complete datasets that are particularly critical for the discovery and validation of targets in hypothesis driven studies in system biology result We introduce new open source software pipeline ATAQS automated and targeted analysis with quantitative SRM which consists of number of modules that collectively support the SRM assay development workflow for targeted proteomic experiments project management and generation of protein peptide and transitions and the validation of peptide detection by SRM ATAQS provides flexible pipeline for end users by allowing the workflow to start or end at any point of the pipeline and for computational biologists by enabling the easy extension of java algorithm classes for their own algorithm plug in or connection via an external web site this integrated system supports all steps in SRM based experiment and provides user friendly GUI that can be run by any operating system that allows the installation of the mozilla firefox web browser conclusions targeted proteomics via SRM is powerful new technique that enables the reproducible and accurate identification and quantification of sets of protein of interest ATAQS is the first open source software that supports all steps of the targeted proteomics workflow ATAQS also provides software API application program interface documentation that enables the addition of new algorithms to each of the workflow steps the software installation guide and sample dataset can be found in http tools proteomecenter org ATAQS ATAQS html

phospopep version is project to support system biology signaling research by providing interactive interrogation of MS derived phosphorylation data from four different organisms currently the database hosts phosphorylation data from the fly drosophila melanogaster human homo sapiens worm caenorhabditis elegans and yeast saccharomyces cerevisiae the following will give an overview of the content and usage of the phosphopep database

the evaluation of biomarkers in bodily fluids necessitates the development of robust method to quantify protein in complex background using large sets of samples the ability to multiplex numerous analytes in single assay expedites the process liquid chromatography mass spectrometry LC MS analyses performed in selected reaction monitoring SRM in conjunction with stable isotope dilution MS present an effective way to detect and quantify biomarker candidates in bodily fluids the strategy presented involves an initial qualification of predefined sets of protein in urine the technique was applied to detect and quantify peptide in urine samples as surrogates for few endogenous protein multiplexed assay were developed to analyze protein associated with bladder cancer few exogenous protein were added as internal standards the sample preparation and the analytical protocols were optimized to ensure reproducibility analytical precision and quantification limits in the low nanogram per milliliter range analyses were performed using known amounts of isotopically labeled peptide systematic replication of the measurements indicated intra assay and inter assay variability with CVs in the range of the differences measured for two targeted protein were correlated with their level of expression in the corresponding tumors using immunohistochemistry

comprehensive characterization of proteome defines fundamental goal in proteomics In order to maximize proteome coverage for complex protein mixture to identify as many protein as possible various different fractionation experiments are typically performed and the individual fractions are subjected to mass spectrometric analysis the resulting data are integrated into large and heterogeneous datasets proteome coverage prediction refers to the task of extrapolating the number of protein discoveries by future measurements conditioned on sequence of already performed measurements proteome coverage prediction at an early stage enables experimentalists to design and plan efficient proteomics studies To date there does not exist any method that reliably predicts proteome coverage from integrated datasets We present generalized hierarchical pitman yor process model that explicitly captures the redundancy within integrated datasets the accuracy of our approach for proteome coverage prediction is assessed by applying it to an integrated proteomics dataset for the bacterium interrogans the proposed procedure outperforms ad hoc extrapolation method and prediction method designed for non integrated datasets furthermore the maximally achievable proteome coverage is estimated for the experimental setup underlying the interrogans dataset We discuss the implications of our results for determining rational stop criteria and their influence on the design of efficient and reliable proteomics studies

biomedical research requires protein detection technology that is not only sensitive and quantitative but that can reproducibly measure any set of protein in biological system in high throughput manner here we report the development and application of targeted proteomics platform termed index ion triggered MS2 ion quantification iMSTIQ that allows reproducible and accurate peptide quantification in complex mixtures the key feature of iMSTIQ is an approach called index ion triggered analysis ITA that permits the reproducible acquisition of full MS2 spectra of targeted peptide independent of their ion intensities accurate quantification is achieved by comparing the relative intensities of multiple pairs of fragment ions derived from isobaric targeted peptide during MS2 analysis importantly the method takes advantage of the favorable performance characteristics of the LTQ orbitrap which include high mass accuracy resolution and throughput As such it provides an attractive targeted proteomics tool to meet the demands of system biology research and biomarker studies

A key barrier to the realization of personalized medicine for cancer is the identification of biomarkers here we describe two stage strategy for the discovery of serum biomarker signatures corresponding to specific cancer causing mutations and its application to prostate cancer PCa in the context of the commonly occurring phosphatase and tensin homolog PTEN tumor suppressor gene inactivation In the first stage of our approach we identified linked glycoproteins from sera and prostate tissue of wild type and pten null mice using label free quantitative proteomics we showed that pten inactivation leads to measurable perturbations in the murine prostate and serum glycoproteome following bioinformatic prioritization in second stage we applied targeted proteomics to detect and quantify human ortholog candidate biomarkers in the sera of PCa patients and control individuals the resulting proteomic profiles were analyzed by machine learning to build predictive regression models for tissue PTEN status and diagnosis and grading of PCa our approach suggests general path to rational cancer biomarker discovery and initial validation guided by cancer genetics and based on the integration of experimental mouse models proteomics based technologies and computational modeling

disassembly of nuclear pore complexes NPCs is decisive event during mitotic entry in cell undergoing open mitosis yet the molecular mechanisms underlying NPC disassembly are unknown using chemical inhibition and depletion experiments we show that NPC disassembly is phosphorylation driven process dependent on CDK1 activity and supported by members of the NIMA related kinase nek family We identify phosphorylation of the GLFG repeat nucleoporin nup98 as an important step in mitotic NPC disassembly mitotic hyperphosphorylation of nup98 is accomplished by multiple kinases including CDK1 and neks nuclei carrying phosphodeficient mutant of nup98 undergo nuclear envelope breakdown slowly such that both the dissociation of nup98 from NPCs and the permeabilization of the nuclear envelope are delayed together our data provide evidence for phosphorylation dependent mechanism underlying disintegration of NPCs during prophase moreover we identify mitotic phosphorylation of nup98 as rate limiting step in mitotic NPC disassembly

decades of biochemical research have identified most of the enzymes that catalyze metabolic reactions in the yeast saccharomyces cerevisiae the adaptation of metabolism to changing nutritional conditions in contrast is much less well understood As an important stepping stone toward such understanding we exploit the power of proteomics assay based on selected reaction monitoring SRM mass spectrometry to quantify abundance changes of the protein that constitute the central carbon and amino-acid metabolic network in the yeast saccharomyces cerevisiae at five different metabolic steady states overall of the targeted protein including families of isoenzymes were consistently detected and quantified in each sample generating proteomic data set that represents nutritionally perturbed biological system at high reproducibility the data set is near comprehensive because we detect of all protein that are required under given condition interpreted through flux balance modeling the data indicate that cerevisiae retains protein not necessarily used in particular environment further the data suggest differential functionality for several metabolic isoenzymes

proteomics is gradually complementing large shotgun qualitative studies with hypothesis driven quantitative experiments targeted analyses performed on triple quadrupole instruments in selected reaction monitoring mode are characterized by high degree of selectivity and low limit of detection however the concurrent analysis of multiple analytes occurs at the expense of sensitivity because of reduced dwell time and or selectivity due to limitation to few transitions new data acquisition paradigm is presented in which selected reaction monitoring is performed in two ways to simultaneously quantify and confirm the identity of the targeted peptide first set of primary transitions is continuously monitored during predetermined elution time window to precisely quantify each peptide In addition set of six to eight transitions is acquired in data dependent event triggered when all the primary transitions exceed preset threshold these additional transitions are used to generate composite tandem mass spectra to formally confirm the identity of the targeted peptide this technique was applied to analyze the tryptic digest of yeast lysate to demonstrate the performance of the technique We showed limit of detection down to tens of attomoles injected and throughput exceeding transitions in one min experiment the technique was integrated into linear work flow including experimental design data acquisition and data evaluation enabling large scale proteomic studies

extensive proteome discovery projects using variety of mass spectrometric techniques have identified protein matching to of the predicted gene models of various species comprehensive proteome coverage is desirable for the unbiased comparison of protein quantities between different biological states and for the meaningful comparison of data from multiple samples here we discuss the feasibility of this goal in the light of recent technological developments

background glucose inhibition of gluconeogenic growth suppressor protein gis2p and zinc finger protein ZNF9 are conserved yeast and human zinc finger protein the function of yeast gis2p is unknown but human ZNF9 has been reported to bind nucleic acids and mutations in the ZNF9 gene cause the neuromuscular disease myotonic dystrophy type To explore the impact of these protein on RNA regulation we undertook systematic analysis of the RNA targets and of the global implications for gene expression results hundreds of mRNAs were associated with gis2p mainly coding for RNA processing factors chromatin modifiers and GTpases target mRNAs contained stretches of trinucleotide repeats located in coding sequence which are sufficient for binding to both gis2p and ZNF9 thus implying strong structural conservation predicted ZNF9 targets belong to the same functional categories as seen in yeast indicating functional conservation which is further supported by complementation of the large cell size phenotype of gis2 mutants with ZNF9 We further applied matched sample proteome transcriptome analysis suggesting that gis2p differentially coordinates expression of RNA regulons primarily by reducing mRNA and protein levels of gene required for ribosome assembly and by selectively up regulating protein levels of myosins conclusions this integrated systematic exploration of RNA targets for homologous RNA binding protein indicates an unexpectedly high conservation of the RNA binding properties and of potential targets thus predicting conserved RNA regulons We also predict regulation of muscle specific gene by ZNF9 adding potential link to the myotonic dystrophy related phenotypes seen in ZNF9 mouse models

the development of plasma biomarkers has proven to be more challenging than initially anticipated many studies have reported lists of candidate protein rather than validated candidate markers with an assigned performance to specific clinical objective biomarker research necessitates clear rational framework with requirements on multitude of levels onthe technological front the platform needs to be effective to detect low abundant plasma protein and be able to measure them in high throughput manner over large amount of samples reproducibly At conceptual level the choice of the technological platform and available samples should be part of an overall clinical study design that depends on joint effort between basic and clinical research solutions to these needs are likely to facilitate more feasible studies targeted proteomic workflows based on SRM mass spectrometry show the potential of fast verification of biomarker candidates in plasma and thereby closing the gap between discovery and validation in the biomarker development pipeline biological samples need to be carefully chosen based on well established guidelines either for candidate discovery in the form of disease models with optimal fidelity to human disease or for candidate evaluation as well designed and annotated clinical cohort groups most importantly they should be representative of the target population and directly address the investigated clinical question conceptual structure of biomarker study can be provided in the form of several sequential phases each having clear objectives and predefined goals furthermore guidelines for reporting the outcome of biomarker studies are critical to adequately assess the quality of the research interpretation and generalization of the results By being attentive to and applying these considerations biomarker research should become more efficient and lead to directly translatable biomarker candidates into clinical evaluation

the protein information and property explorer PIPE2 is an enhanced software program and updated web application that aims at providing the proteomic researcher simple intuitive user interface through which to begin inquiry into the biological significance of list of protein typically produced by MS MS proteomic processing software PIPE2 includes an improved interface new data visualization options and new data analysis method for combining disparate but related data sets In particular PIPE2 has been enhanced to handle multi dimensional data such as protein abundance gene expression and or interaction data the current architecture of PIPE2 modeled after that of gaggle programming infrastructure for interoperability between separately developed software tools contains independent functional units that can be instantiated and pieced together at the user discretion to form pipelined analysis workflow among these functional units is the network viewer component which adds rich network analysis capabilities to the suite of existing proteomic web resources additionally PIPE2 implements framework within which new analysis procedures can be easily deployed and distributed over the world wide web PIPE2 is available as web service at

glycosylation is one of the most important and common forms of protein post translational modification that is involved in many physiological functions and biological pathways altered glycosylation has been associated with variety of diseases including cancer inflammatory and degenerative diseases glycoproteins are becoming important targets for the development of biomarkers for disease diagnosis prognosis and therapeutic response to drugs the emerging technology of glycoproteomics which focuses on glycoproteome analysis is increasingly becoming an important tool for biomarker discovery An in depth comprehensive identification of aberrant glycoproteins and further quantitative detection of specific glycosylation abnormalities in complex environment require concerted approach drawing from variety of techniques this report provides an overview of the recent advances in mass spectrometry based glycoproteomic method and technology in the context of biomarker discovery and clinical application

comprehensive characterization of proteome defines fundamental goal in proteomics In order to maximize proteome coverage for complex protein mixture to identify as many protein as possible various different fractionation experiments are typically performed and the individual fractions are subjected to mass spectrometric analysis the resulting data are integrated into large and heterogeneous datasets proteome coverage prediction refers to the task of extrapolating the number of protein discoveries by future measurements conditioned on sequence of already performed measurements proteome coverage prediction at an early stage enables experimentalists to design and plan efficient proteomics studies To date there does not exist any method that reliably predicts proteome coverage from integrated datasets We present generalized hierarchical pitman yor process model that explicitly captures the redundancy within integrated datasets We assess the proteome coverage prediction accuracy of our approach applied to an integrated proteomics dataset for the bacterium interrogans and we demonstrate that it outperforms ad hoc extrapolation method and prediction method designed for non integrated datasets furthermore we estimate the maximally achievable proteome coverage for the experimental setup underlying the interrogans dataset We discuss the implications of our results to determine rational stop criteria and their influence on the design of efficient and reliable proteomics studies

this is conversation with bernd bodenmiller and ruedi aebersold about research article published in the december issue of science signaling bodenmiller and aebersold describe the effect that targeted removal of individual kinases and phosphatases had on the yeast phosphoproteome their study revealed changes in phosphoproteins that were not direct substrates of the targeted enzymes and demonstrated that the network of kinases and phosphatases is surprisingly robust

the phosphorylation and dephosphorylation of protein by kinases and phosphatases constitute an essential regulatory network in eukaryotic cell this network supports the flow of information from sensors through signaling system to effector molecules and ultimately drives the phenotype and function of cell tissues and organisms dysregulation of this process has severe consequencesand is one of the main factors in the emergence and progression of diseases including cancer thus major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases however it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as whole here we used label free quantitative phosphoproteomics in systematically perturbed model organism saccharomyces cerevisiae to determine the relationships between kinases phosphatases and more than phosphoproteins We identified regulated phosphorylation events describing the first system wide protein phosphorylation network in vivo our results show that at steady state inactivation of most kinases and phosphatases affected large parts of the phosphorylation modulated signal transduction machinery and not only the immediate downstream targets the observed cellular growth phenotype was often well maintained despite the perturbations arguing for considerable robustness in the system our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis

the double strand break DSB is cytotoxic DNA lesion caused by oxygen radicals ionizing radiation and radiomimetic chemicals cell cope with DNA damage by activating the DNA damage response DDR which leads either to damage repair and cellular survival or to programmed cell death the main transducer of the DSB response is the nuclear protein kinase ataxia telangiectasia mutated ATM We applied label free quantitative mass spectrometry to follow the dynamics of DSB induced phosphoproteome in nuclear fractions of the human melanoma G361 cell after radiomimetic treatment We found that these dynamics are complex including both phosphorylation and dephosphorylation events In addition to identifying previously unknown ATM dependent phosphorylation and dephosphorylation events we found that about of DSB induced phosphorylations were ATM independent and that several other kinases are potentially involved sustained activity of ATM was required to maintain many ATM dependent phosphorylations We identified an ATM dependent phosphorylation site on ATM itself that played role in its retention on damaged chromatin By connecting many of the phosphorylated and dephosphorylated protein into functional networks we highlight putative cross talks between protein pertaining to several cellular biological process our study expands the DDR phosphorylation landscape and identifies previously unknown ATM dependent and independent branches It reveals insights into the breadth and complexity of the cellular responses involved in the coordination of many DDR pathways which is in line with the critical importance of genomic stability in maintenance of cellular homeostasis

the structure of the 26S proteasome from schizosaccharomyces pombe has been determined to resolution of by cryoelectron microscopy and single particle analysis In addition chemical cross linking in conjunction with mass spectrometry has been used to identify numerous residue pairs in close proximity to each other providing an array of spatial restraints taken together these data clarify the topology of the AAA ATpase module in the 19S regulatory particle and its spatial relationship to the ring of the 20S core particle image classification and variance analysis reveal belt of high activity surrounding the AAA ATpase module which is tentatively assigned to the reversible association of proteasome interacting protein and the conformational heterogeneity among the particles An integrated model is presented which sheds light on the early steps of protein degradation by the 26S complex

although cellular behaviors are dynamic the networks that govern these behaviors have been mapped primarily as static snapshots using an approach called differential epistasis mapping we have discovered widespread changes in genetic interaction among yeast kinases phosphatases and transcription factors as the cell responds to DNA damage differential interactions uncover many gene functions that go undetected in static conditions they are very effective at identifying DNA repair pathways highlighting new damage dependent roles for the slt2 kinase pph3 phosphatase and histone variant htz1 the data also reveal that protein complexes are generally stable in response to perturbation but the functional relations between these complexes are substantially reorganized differential networks chart new type of genetic landscape that is invaluable for mapping cellular responses to stimuli

forkhead transcription factors of the foxO subfamily regulate gene expression programs downstream of the insulin signaling network It is less clear which protein mediate transcriptional control exerted by target of rapamycin TOR signaling but recent studies in nematodes suggest role for foxA transcription factors downstream of TOR In this study we present evidence that outlines similar connection in drosophila in which the foxA protein fork head FKH regulates cellular and organismal size downstream of TOR We find that ectopic expression and targeted knockdown of FKH in larval tissues elicits different size phenotypes depending on nutrient state and TOR signaling levels FKH overexpression has negative effect on growth under fed conditions and this phenotype is not further exacerbated by inhibition of TOR via rapamycin feeding under conditions of starvation or low TOR signaling levels knockdown of FKH attenuates the size reduction associated with these conditions subcellular localization of endogenous FKH protein is shifted from predominantly cytoplasmic on high protein diet to pronounced nuclear accumulation in animals with reduced levels of TOR or fed with rapamycin two putative FKH target gene CG6770 and cabut are transcriptionally induced by rapamycin or FKH expression and silenced by FKH knockdown induction of both target gene in heterozygous TOR mutant animals is suppressed by mutations in fkh furthermore TOR signaling levels and FKH impact on transcription of the dFOXO target gene d4E BP implying point of crosstalk with the insulin pathway In summary our observations show that an alteration of FKH levels has an effect on cellular and organismal size and that FKH function is required for the growth inhibition and target gene induction caused by low TOR signaling levels

proteomes the ensembles of all protein expressed by cell or tissues are typically analysed by mass spectrometry recent technical and computational advances have greatly increased the fraction of proteome that can be identified and quantified in single study current mass spectrometry based proteomic strategies have the potential to reproducibly accurately quantitatively and comprehensively measure any protein or whole proteomes from cell and tissues at different states achieving these goals will require complete proteome maps and analytical strategies that use these maps as prior information and will greatly enhance the impact of proteomics on biological and clinical research

protein ubiquitination is an essential post translational modification PTM involved in the regulation of variety of cellular functions including transcription and protein degradation protein can be both mono or poly ubiquitinated poly ubiquitin chains vary in the manner by which the ubiquitin protein are linked and their total length different poly ubiquitin structures are thought to specify different fates for the target protein but the correlation between poly ubiquitin structures and their specific cellular function is not well understood We have developed set of specific and quantitative targeted mass spectrometry assay to determine the frequency of different types of inter ubiquitin linkages in poly ubiquitin chains relative to the total ubiquitin concentration We chemically synthesized heavy isotope labeled reference peptide that represent the products generated by tryptic digestion of the known forms of inter ubiquitin links for the yeast saccharomyces cerevisiae and human in addition to all peptide from tryptic digestion of single ubiquitin molecule for these two species We used these peptide to develop optimized selected reaction monitoring SRM assay for their unambiguous detection in biological samples We used these assay to profile the frequency of the different types of inter ubiquitin linkages in mixture of in vitro assembled human poly ubiquitin chains and isolated poly ubiquitinated protein from cerevisiae We then applied the method to detect toxin induced changes in the poly ubiquitination profile in complex and enriched protein samples

efficient experimental strategies are needed to validate computationally predicted microRNA miRNA target gene here we present large scale targeted proteomics approach to validate predicted miRNA targets in caenorhabditis elegans using selected reaction monitoring SRM we quantified protein of interest in extracts from wild type and let mutant worms We demonstrate by independent experimental downstream analyses such as genetic interaction as well as polysomal profiling and luciferase assay that validation by targeted proteomics substantially enriched for biologically relevant let interactors for example we found that the zinc finger protein ZTF was bona fide let miRNA target We also validated predicted miR targets demonstrating that this approach is adaptable to other miRNAs We propose that targeted mass spectrometry can be applied generally to validate candidate lists generated by computational method or in large scale experiments and that the described strategy should be readily adaptable to other organisms

cellular functions are largely carried out by noncovalent protein complexes that may exist within the cell as stable modules or as assemblies of dynamically changing composition whose formation and decomposition are triggered in response to extracellular stimuli the protein constituents of complexes often exhibit post translational modifications such as phosphorylation that can impact their ability to interact with other protein and thus to form multicomponent complexes complete characterization of particular protein complex thus requires determining both the identity of interacting protein and their covalent modifications in terms of attachment sites and stoichiometry We have previously developed protocol which identifies genuine constituents of partially purified protein complexes and concurrently determines their phosphorylation sites and levels in single LC MS MS analysis performed on MALDI TOF TOF instrument pflieger junger muller rinner lee gehrig gstaiger aebersold mol cell proteomics the method combines fourplex iTRAQ labeling isobaric tags for relative and absolute quantification and phosphatase treatment of peptide samples derived from the tryptic digestion of isolated complexes To test the performances of this method with nanoESI and different peptide fragmentation modes possibly better suited for the identification of phosphorylated sequence than MALDI TOF TOF MS we have implemented it on the nanoESI LTQ orbitrap instrument the model protein beta casein was used to optimize the conditions with respect to sensitivity and quantitative accuracy combination of CID fragmentation in the linear ion trap and higher energy collision dissociation HCD appeared optimal to obtain reliable and robust identification and quantification data the optimized conditions were then applied to identify and estimate the respective levels of phosphorylation sites on the purified autoactivated tyrosine kinase domain of fibroblast growth factor receptor FGFR3 KD and to analyze complexes formed around the insulin receptor substrate homologue CHICO immunopurified from drosophila melanogaster cell that were either stimulated with insulin or left untreated these new analyses allowed us to improve the assignment of the phosphorylation sites of some peptide previously detected by MALDI TOF TOF analysis and to identify additional phosphorylated sequence in CHICO and in the insulin receptor

immunophenotyping by flow cytometry or immunohistochemistry is clinical standard procedure for diagnosis classification and monitoring of hematologic malignancies antibody based cell surface phenotyping is commonly limited to cell surface protein for which specific antibodies are available and the number of parallel measurements is limited the resulting limited knowledge about cell surface protein markers hampers early clinical diagnosis and subclassification of hematologic malignancies here we describe the mass spectrometry based phenotyping of all trans retinoic acid treated acute myeloid leukemia model system at an unprecedented level to depth of more than membrane protein including bona fide cell surface exposed CD protein this extensive view of the leukemia surface proteome was achieved by developing and applying new implementations of the cell surface capturing CSC technology bioinformatic and hierarchical cluster analysis showed that the applied strategy reliably revealed known differentiation induced abundance changes of cell surface protein in HL60 and NB4 cell and it also identified cell surface protein with very little prior information the extensive and quantitative analysis of the cell surface protein landscape from system biology perspective will be most useful in the clinic for the improved subclassification of hematologic malignancies and the identification of new drug targets

spectral library searching is an emerging approach in proteomic data analysis for the inference of peptide identifications from tandem mass spectra It offers promising alternative to sequence database searching currently the dominant method for this purpose In spectral searching spectral library is first meticulously compiled from large collection of previously observed and identified peptide MS MS spectra the spectrum of the unknown peptide can then by identified by comparing it to all the candidates in the spectral library for the best match this unit covers the basic principles of spectral searching describes its advantages and limitations and reviews the available software tools developed for spectral library searching and building in terms of their algorithms and their surrounding informatics support

systems biology at the molecular level is concerned with networks of interacting molecules their structure and dynamic response to perturbations that give rise to system properties that determine measurable macroscopic phenotypes At any time in any cell multiple types of molecular networks are concurrently active one of the most important known regulatory system in eukaryotic cell is reversible protein phosphorylation catalyzed by protein kinases and phosphatases respectively therefore it is essential to understand and eventually model the protein phosphorylation mediated informational fluxes in cell from sensors and signaling system to effector molecules to comprehensively analyze the dynamic system of kinases phosphatases and their substrates and to determine the basic rules of information processing in cell In this chapter we describe the protocols necessary to comprehensively and quantitatively measure the phosphorylation modulated informational networks in cell the pipeline relies on the selective quantitative isolation of phosphopeptides generated by the tryptic digestion of complex protein mixtures and their subsequent mass spectrometric and computational analysis We believe that the protocols and data processing tools described in this chapter will be valuable resource for biologists interested in the analysis of protein phosphorylation based signal transduction

chemical cross linking of reactive groups in native protein and protein complexes in combination with the identification of cross linked sites by mass spectrometry has been in use for more than decade recent advances in instrumentation cross linking protocols and analysis software have led to renewed interest in this technique which promises to provide important information about native protein structure and the topology of protein complexes In this article we discuss the critical steps of chemical cross linking and its implications for structural biology reagent design and cross linking protocols separation and mass spectrometric analysis of cross linked samples dedicated software for data analysis and the use of cross linking data for computational modeling finally the impact of protein cross linking on various biological disciplines is highlighted

the vast majority of proteomic studies to date have relied on mass spectrometric techniques to identify and in some cases quantify peptide that have been generated by proteolysis current approaches differ in the types of instrument used their performance profiles the manner in which they interface with biological research strategies and their reliance on and use of prior information here we consider the three main mass spectrometry MS based proteomic approaches used today shotgun or discovery directed and targeted strategies We discuss the principles of each technique their strengths and weaknesses and the dependence of their performance profiles on the composition of the biological sample our goal is to provide rational framework for selecting strategies optimally suited to address the specific research issue under consideration

under aerobic high glucose conditions saccharomyces cerevisiae exhibits glucose repression and thus predominantly fermentative metabolism here we show that two commonly used prototrophic representatives of the CEN PK and S288C strain families respond differently to deletion of the hexokinase HXK2 key player in glucose repression In CEN PK growth rate collapses and derepression occurs on the physiological level while the S288C descendant FY4 hxk2 still grows like the parent strain and shows fully repressed metabolism CEN PK hxk2 strain with repaired adenylate cyclase gene CYR1 maintains repression but not growth rate comparison of the parent strain physiology metabolome and proteome revealed higher metabolic rates identical biomass and byproduct yields suggesting lower snf1 activity and higher protein kinase PKA activity in CEN PK this study highlights the importance of the genetic background in the process of glucose signaling and regulation contributes novel evidence on the overlap between the classical glucose repression pathway and the cAMP PKA signaling pathway and might have the potential to resolve some of the conflicting findings existing in the field

spectral library searching is an emerging approach in proteomic data analysis for the inference of peptide identifications from tandem mass spectra It offers promising alternative to sequence database searching currently the dominant method for this purpose In spectral searching spectral library is first meticulously compiled from large collection of previously observed and identified peptide MS MS spectra the spectrum of the unknown peptide can then by identified by comparing it to all the candidates in the spectral library for the best match this unit covers the basic principles of spectral searching describes its advantages and limitations and reviews the available software tools developed for spectral library searching and building in terms of their algorithms and their surrounding informatics support

spectral library searching is new approach in proteomic data analysis that promises to address some of the shortcomings of sequence database searching currently the dominant method for inferring peptide identifications from tandem mass spectra In spectral searching spectral library is first meticulously compiled from large collection of previously observed and identified peptide MS MS spectra the unknown spectrum can then be identified by comparing it to all the candidates in the spectral library for the best match It offers the benefits of tremendous speed gain and increase in sensitivity and selectivity compared to sequence searching this article provides concise roadmap for the proteomics researchers to start using spectral library searching in their data analysis workflow

the trans proteomic pipeline TPP is suite of software tools for the analysis of MS MS data sets the tools encompass most of the steps in proteomic data analysis workflow in single integrated software system specifically the TPP supports all steps from spectrometer output file conversion to protein level statistical validation including quantification by stable isotope ratios We describe here the full workflow of the TPP and the tools therein along with an example on sample data set demonstrating that the setup and use of the tools are straightforward and well supported and do not require specialized informatic resources or knowledge

electron transfer dissociation ETD is an alternative fragmentation technique to CID that has recently become commercially available ETD has several advantages over CID It is less prone to fragmenting amino-acid side chains especially those that are modified thus yielding fragment ion spectra with more uniform peak intensities further precursor ions of longer peptide and higher charge states can be fragmented and identified however analysis of ETD spectra has few important differences that require the optimization of the software packages used for the analysis of CID data or the development of specialized tools We have adapted the trans proteomic pipeline to process ETD data specifically we have added support for fragment ion spectra from high charge precursors compatibility with charge state estimation algorithms provisions for the use of the lys protease capabilities for ETD spectrum library building and updates to the data formats to differentiate CID and ETD spectra We show the results of processing data sets from several different types of ETD instruments and demonstrate that application of the ETD enhanced trans proteomic pipeline can increase the number of spectrum identifications at fixed false discovery rate by as much as over native output from single sequence search engine

despite the essential roles of sphingolipids both as structural components of membranes and critical signalling molecules we have limited understanding of how cell sense and regulate their levels here we reveal the function in sphingolipid metabolism of the ORM gene known as ORMDL gene in humans conserved gene family that includes ORMDL3 which has recently been identified as potential risk factor for childhood asthma starting from an unbiased functional genomic approach in saccharomyces cerevisiae we identify orm protein as negative regulators of sphingolipid synthesis that form conserved complex with serine palmitoyltransferase the first and rate limiting enzyme in sphingolipid production We also define regulatory pathway in which phosphorylation of orm protein relieves their inhibitory activity when sphingolipid production is disrupted changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism our work identifies the orm protein as critical mediators of sphingolipid homeostasis and raises the possibility that sphingolipid misregulation contributes to the development of childhood asthma

the challenge of estimating false discovery rates FDR in peptide identification from MS MS spectra has received increased attention in proteomics the simple approach of target decoy searching has become popular with traditional sequence database searching method but has yet to be practiced in spectral library searching an emerging alternative to sequence searching We extended this target decoy searching approach to spectral searching by developing and validating robust method to generate realistic but unnatural decoy spectra our method involves randomly shuffling the peptide identification of each reference spectrum in the library and repositioning each fragment ion peak along the axis to match the fragment ions expected from the shuffled sequence We show that this method produces decoy spectra that are sufficiently realistic such that incorrect identifications are equally likely to match real and decoy spectra key assumption necessary for decoy counting this approach has been implemented in the open source library building software spectraST

selected reaction monitoring SRM uses sensitive and specific mass spectrometric assay to measure target analytes across multiple samples but it has not been broadly applied in proteomics owing to the tedious assay development process for each protein We describe method based on crude synthetic peptide libraries for the high throughput development of SRM assay We illustrate the power of the approach by generating and applying validated SRM assay for all saccharomyces cerevisiae kinases and phosphatases

comprehensive characterization of proteome is fundamental goal in proteomics To achieve saturation coverage of proteome or specific subproteome via tandem mass spectrometric identification of tryptic protein sample digests proteomics data sets are growing dramatically in size and heterogeneity the trend toward very large integrated data sets poses so far unsolved challenges to control the uncertainty of protein identifications going beyond well established confidence measures for peptide spectrum matches We present MAYU novel strategy that reliably estimates false discovery rates for protein identifications in large scale data sets We validated and applied MAYU using various large proteomics data sets the data show that the size of the data set has an important and previously underestimated impact on the reliability of protein identifications We particularly found that protein false discovery rates are significantly elevated compared with those of peptide spectrum matches the function provided by MAYU is critical to control the quality of proteome data repositories and thereby to enhance any study relying on these data sources the MAYU software is available as standalone software and also integrated into the trans proteomic pipeline

As the application for quantitative proteomics in the life sciences has grown in recent years so has the need for more robust and generally applicable method for quality control and calibration the reliability of quantitative proteomics is tightly linked to the reproducibility and stability of the analytical platforms which are typically multicomponent sample preparation multistep separations and mass spectrometry with individual components contributing unequally to the overall system reproducibility variations in quantitative accuracy are thus inevitable and quality control and calibration become essential for the assessment of the quality of the analyses themselves toward this end the use of internal standards cannot only assist in the detection and removal of outlier data acquired by an irreproducible system quality control but can also be used for detection of changes in instruments for their subsequent performance and calibration here we introduce set of halogenated peptide as internal standards the peptide are custom designed to have properties suitable for various quality control assessments data calibration and normalization process the unique isotope distribution of halogenated peptide makes their mass spectral detection easy and unambiguous when spiked into complex peptide mixtures In addition they were designed to elute sequentially over an entire aqueous to organic LC gradient and to have values within the commonly scanned mass range Da In series of experiments in which these peptide were spiked into an enriched glycosite peptide fraction from formerly glycosylated intact protein in their deglycosylated form isolated from human plasma we show the utility and performance of these halogenated peptide for sample preparation and LC injection quality control as well as for retention time and mass calibration further use of the peptide for signal intensity normalization and retention time synchronization for selected reaction monitoring experiments is also demonstrated

patients with ulcerative colitis UC have an increased risk for developing colorectal cancer because UC tumorigenesis is associated with genomic field defects that can extend throughout the entire colon including the non dysplastic mucosa we hypothesized that the same field defects will include abnormally expressed protein here we applied proteomics to study the protein expression of UC neoplastic progression the protein profiles of colonic epithelium were compared with UC patients without dysplasia non progressors ii nondysplastic colonic tissue from UC patient with high grade dysplasia or cancer progressors iii high grade dysplastic tissue from UC progressors and iv normal colon We identified differential protein expression associated with UC neoplastic progression protein relating to mitochondria oxidative activity and calcium binding protein were some of the interesting classes of these protein network analysis discovered that Sp1 and myc protein may play roles in UC early and late stages of neoplastic progression respectively two over expressed protein in the non dysplastic tissue of UC progressors carbamoyl phosphate synthase and S100P were further confirmed by immunohistochemistry analysis our study provides insight into the molecular events associated with UC neoplastic progression which could be exploited for the development of protein biomarkers in fields of non dysplastic mucosa that identify patient risk for UC dysplasia

To date the vast majority of the proteomic data sets collected by mass spectrometry MS have been generated by nondirected method whereby the identified precursor ions are stochastically selected for sequencing from complex sample mixtures recently new MS approaches have been developed in which the mass spectrometer is directed to select and fragment sets of precursor ions that represent the most informative peptide in sample mixture these directed MS method have shown superior performance for the fast sensitive and highly reproducible generation of consistent data sets at low redundancy In this manuscript we summarize recent technical advances in directed MS and discuss important applications to quantitative proteomics

the identification of specific biomarkers will improve the early diagnosis of disease facilitate the development of targeted therapies and provide an accurate method to monitor treatment response major challenge in the process of verifying biomarker candidates in blood plasma is the complexity and high dynamic range of protein this article reviews the current targeted proteomic strategies that are capable of quantifying biomarker candidates at concentration ranges where biomarkers are expected in plasma at the ng ml level In addition workflow is presented that allows the fast and definitive generation of targeted mass spectrometry based assay for most biomarker candidate protein these assay are stored in publicly accessible databases and have the potential to greatly impact the throughput of biomarker verification studies

the genome of mycoplasma pneumoniae is among the smallest found in self replicating organisms To study the basic principles of bacterial proteome organization we used tandem affinity purification mass spectrometry TAP MS in proteome wide screen the analysis revealed homomultimeric and heteromultimeric soluble protein complexes of which the majority are novel about third of the heteromultimeric complexes show higher levels of proteome organization including assembly into larger multiprotein complex entities suggesting sequential steps in biological process and extensive sharing of components implying protein multifunctionality incorporation of structural models for protein single particle electron microscopy and cellular electron tomograms provided supporting structural details for this proteome organization the data set provides blueprint of the minimal cellular machinery required for life copyright by the american association for the advancement of science all rights reserved

protein modifications play major role for most biological process in living organisms amino terminal acetylation of protein is common modification found throughout the tree of life the terminus of nascent polypeptide chain becomes co translationally acetylated often after the removal of the initiating methionine residue while the enzymes and protein complexes involved in these process have been extensively studied only little is known about the biological function of such terminal modification events To identify common principles of terminal acetylation we analyzed the amino terminal peptide from protein extracted from drosophila Kc167 cell We detected more than mature protein termini and could show that terminal acetylation occurs in insects with similar frequency as in humans As the sole true determinant for terminal acetylation we could extract the PX rule that indicates the prevention of acetylation under all circumstances We could show that this rule can be used to genetically engineer protein to study the biological relevance of the presence or absence of an acetyl group thereby generating generic assay to probe the functional importance of terminal acetylation We applied the assay by expressing mutated protein as transgenes in cell lines and in flies here we present straightforward strategy to systematically study the functional relevance of terminal acetylations in cell and whole organisms since the PX rule seems to be of general validity in lower as well as higher eukaryotes we propose that it can be used to study the function of terminal acetylation in all species

systems biology conceptualizes biological system as dynamic networks of interacting elements whereby functionally important properties are thought to emerge from the structure of such networks owing to the ubiquitous role of complexes of interacting protein in biological system their subunit composition and temporal and spatial arrangement within the cell are of particular interest visual proteomics attempts to localize individual macromolecular complexes inside of intact cell by template matching reference structures into cryo electron tomograms here we combined quantitative mass spectrometry and cryo electron tomography to detect count and localize specific protein complexes in the cytoplasm of the human pathogen leptospira interrogans We describe scoring function for visual proteomics and assess its performance and accuracy under realistic conditions We discuss current and general limitations of the approach as well as expected improvements in the future

multiple reaction monitoring mass spectrometry MRM MS is targeted analysis method that has been increasingly viewed as an avenue to explore proteomes with unprecedented sensitivity and throughput We have developed software tool called MaRimba to automate the creation of explicitly defined MRM transition lists required to program triple quadrupole mass spectrometers in such analyses MaRimba creates MRM transition lists from downloaded or custom built spectral libraries restricts output to specified protein or peptide and filters based on precursor peptide and product ion properties MaRimba can also create MRM lists containing corresponding transitions for isotopically heavy peptide for which the precursor and product ions are adjusted according to user specifications this open source application is operated through graphical user interface incorporated into the transproteomic pipeline and it outputs the final MRM list to text file for upload to MS instruments To illustrate the use of MaRimba we used the tool to design and execute an MRM MS experiment in which we targeted the protein of well defined and previously published standard mixture

the target of rapamycin complex TORC1 is an essential multiprotein complex conserved from yeast to humans under favorable growth conditions and in the absence of the macrolide rapamycin TORC1 is active and influences virtually all aspects of cell growth although two direct effectors of yeast TORC1 have been reported tap42 regulator of PP2A phosphatases and sch9 an AGC family kinase the signaling pathways that couple TORC1 to its distal effectors were not well understood To elucidate these pathways we developed and employed quantitative label free mass spectrometry approach analyses of the rapamycin sensitive phosphoproteomes in various genetic backgrounds revealed both documented and novel TORC1 effectors and allowed us to partition phosphorylation events between tap42 and sch9 follow up detailed characterization shows that sch9 regulates RNA polymerases and III the latter via maf1 in addition to translation initiation and the expression of ribosomal protein and ribosome biogenesis gene this demonstrates that sch9 is master regulator of protein synthesis

the systematic and quantitative molecular analysis of mutant organisms that has been pioneered by studies on mutant metabolomes and transcriptomes holds great promise for improving our understanding of how phenotypes emerge unfortunately owing to the limitations of classical biochemical analysis protein have previously been excluded from such studies here we review how technical advances in mass spectrometry based proteomics can be applied to measure changes in protein abundance posttranslational modifications and protein protein interactions in mutants at the scale of the proteome We finally discuss examples that integrate proteomics data with genomic and phenomic information to build network centred models which provide promising route for understanding how phenotypes emerge

the rise of system biology implied growing demand for highly sensitive techniques for the fast and consistent detection and quantification of target sets of protein across multiple samples this is only partly achieved by classical mass spectrometry or affinity based method We applied targeted proteomics approach based on selected reaction monitoring SRM to detect and quantify protein expressed to concentration below copies cell in total cerevisiae digests the detection range can be extended to single digit copies cell and to protein undetected by classical method We illustrate the power of the technique by the consistent and fast measurement of network of protein spanning the entire abundance range over growth time course of cerevisiae transiting through a series of metabolic phases We therefore demonstrate the potential of SRM based proteomics to provide assay for the measurement of any set of protein of interest in yeast at high throughput and quantitative accuracy

mass spectrometry based method for relative proteome quantification have broadly affected life science research however important research directions particularly those involving mathematical modelling and simulation of biological process also critically depend on absolutely quantitative datag that is knowledge of the concentration of the expressed protein as function of cellular state until now absolute protein concentration measurements of considerable fraction of the proteome have only been derived from genetically altered saccharomyces cerevisiae cell technique that is not directly portable from yeast to other species here we present mass spectrometry based strategy to determine the absolute quantity that is the average number of protein copies per cell in cell population for large fraction of the proteome in genetically unperturbed cell applying the technology to the human pathogen leptospira interrogans spirochete responsible for leptospirosis we generated an absolute protein abundance scale for of the mass spectrometry detectable proteome from cell at different states taking advantage of the unique cellular dimensions of interrogans we used cryo electron tomography morphological measurements to verify at the single cell level the average absolute abundance values of selected protein determined by mass spectrometry on population of cell because the strategy is relatively fast and applicable to any cell type we expect that it will become cornerstone of quantitative biology and system biology

In proteomics rapid developments in instrumentation led to the acquisition of increasingly large data sets correspondingly proDaC was founded in as coordination action project within the 6th european union framework programme to support data sharing and community wide data collection the objectives of proDaC were the development of documentation and storage standards setup of standardized data submission pipeline and collection of data ending in march proDaC has delivered comprehensive toolbox of standards and computer programs to achieve these goals

protein kinases enable cellular information processing although numerous human phosphorylation sites and their dynamics have been characterized the evolutionary history and physiological importance ofmany signaling events remain unknown using target phosphoproteomes determined with similar experimental and computational pipeline we investigated the conservation of human phosphorylation events in distantly related model organisms fly worm and yeast with sequence alignment approach we identified phosphorylation events in human protein that appear to be positionally conserved over million years of evolution and hence are likely to be involved in fundamental cellular process this sequencealignment analysis suggested that many phosphorylation sites evolve rapidly and therefore do not display strong evolutionary conservation in terms of sequence position in distantly related organisms thus we devised network alignment approach to reconstruct conserved kinase substrate networks which identified phosphorylation events in human protein both method identified protein tightly regulated by phosphorylation as well as signal integration hubs and both types of phosphoproteins were enriched in protein encoded by disease associated gene We analyzed the cellular functions and structural relationships for these conserved signaling events noting the incomplete nature of current phosphoproteomes assessing phosphorylation conservation at both site and network levels proved useful for exploring both fast evolving and ancient signaling events We reveal that multiple complex diseases seem to converge within the conserved networks suggesting that disease development might rely on common molecular networks copyright by the american association for the advancement of science all rights reserved

We have developed proteome database DB biomarkerdigger http biomarkerdigger org that automates data analysis searching and metadata gathering function the metadatagathering function searches proteome DBs for protein protein interaction gene ontology protein domain online mendelian inheritance in man and tissue expression profile information and integrates it into protein data sets that are accessed through search function in biomarkerdigger this DB also facilitates cross proteome comparisons by classifying protein based on their annotation biomarkerdigger highlights relationships between given protein in proteomic data set and any known biomarkers or biomarker candidates the newly developed biomarkerdigger system is useful for multi level synthesis comparison and analyses of data sets obtained from currently available web sources We demonstrate the application of this resource to the identification of serological biomarker for hepatocellular carcinoma by comparison of plasma and tissue proteomic data sets from healthy volunteers and cancer patients

motivation liquid chromatography tandem mass spectrometry LC MS MS is the predominant method to comprehensively characterize complex protein mixtures such as samples from prefractionated or complete proteomes In order to maximize proteome coverage for the studied sample identify as many traceable protein as possible LC MS MS experiments are typically repeated extensively and the results combined proteome coverage prediction is the task of estimating the number of peptide discoveries of future LC MS MS experiments proteome coverage prediction is important to enhance the design of efficient proteomics studies To date there does not exist any method to reliably estimate the increase of proteome coverage at an early stage results We propose an extended infinite markov model dirisim to extrapolate the progression of proteome coverage based on small number of already performed LC MS MS experiments the method explicitly accounts for the uncertainty of peptide identifications We tested dirisim on set of LC MS MS experiments of complete proteome sample and demonstrated that dirisim correctly predicts the coverage progression already from small subset of experiments the predicted progression enabled us to specify maximal coverage for the test sample We demonstrated that quality requirements on the final proteome map impose an upper bound on the number of useful experiment repetitions and limit the achievable proteome coverage

better understanding how cell are regulated and adapt to their environment based on the reversible phosphorylation of protein is key question of current molecular and system biology research In this study an advanced mass spectrometry based approach leveraging the electron transfer dissociation ETD technique in combination with CID using linear ion trap mass spectrometer is described the technique was applied for the first time to the identification of phosphorylated peptide isolated from the drosophila melanogaster Kc167 cell line We demonstrate that the method is particularly useful for the characterization of large phosphopeptides including those with multiple phosphorylation sites as extensive series of and fragment ions were observed finally we have applied directed tandem mass spectrometric workflow using inclusion lists to increase the number of identified peptide

patients with pancreatic cancer are usually diagnosed at late stages when the disease is incurable pancreatic intraepithelial neoplasia panIN is believed to be the immediate precursor lesion of pancreatic adenocarcinoma and would be an ideal stage to diagnose patients when intervention and cure are possible and patients are curable In this study we used quantitative proteomics to identify dysregulated protein in panIN lesions altogether over dysregulated protein were identified in the panIN tissues with minimum of fold change compared with the protein in normal pancreas these dysregulated panIN protein play roles in cell motility the inflammatory response the blood clotting cascade the cell cycle and its regulation and protein degradation further network analysis of the protein identified MYC as an important regulatory protein in panIN lesions finally three of the overexpressed protein laminin beta galectin and actinin were validated by immunohistochemistry analysis all three of these protein were overexpressed in the stroma or ductal epithelial cell of advanced panIN lesions as well as in pancreatic cancer tissue our findings suggest that these three protein may be useful as biomarkers for advanced panIN and pancreatic cancer if further validated the dysregulated protein identified in this study may assist in the selection of candidates for future development of biomarkers for detecting early and curable pancreatic neoplasia

proliferation of mammalian cell requires the coordinated function of many protein to accurately divide cell into two daughter cell several RNAi screens have identified previously uncharacterised gene that are implicated in mammalian cell division the molecular function for these gene needs to be investigated to place them into pathways phenotypic profiling is useful method to assign putative functions to uncharacterised gene here we show that the analysis of protein localisation is useful to refine phenotypic profile We show the utility of this approach by defining function of the previously uncharacterised gene C13orf3 during cell division C13orf3 localises to centrosomes the mitotic spindle kinetochores spindle midzone and the cleavage furrow during cell division and is specifically phosphorylated during mitosis furthermore C13orf3 is required for centrosome integrity and anaphase onset depletion by RNAi leads to mitotic arrest in metaphase with an activation of the spindle assembly checkpoint and loss of sister chromatid cohesion proteomic analyses identify C13orf3 ska3 as new component of the ska complex and show direct interaction with regulatory subunit of the protein phosphatase PP2A all together these data identify C13orf3 as an important factor for metaphase to anaphase progression and highlight the potential of combined RNAi screening and protein localisation analyses

the lack of sensitive specific multiplexage assay for most human protein is the major technical barrier impeding development of candidate biomarkers into clinically useful tests recent progress in mass spectrometry based assay for proteotypic peptide particularly those with specific affinity peptide enrichment offers systematic and economical path to comprehensive quantitative coverage of the human proteome complete suite of assay two peptide from the protein product of each of the human gene here termed the human proteome detection and quantitation project would enable rapid and systematic verification of candidate biomarkers and lay quantitative foundation for subsequent efforts to define the larger universe of splice variants post translational modifications protein protein interactions and tissue localization

most protein are post translationally modified and the characterization of modified peptide in complex mixtures generated by enzymatic digestion of multiple protein remains major analytical challenge We describe an integrated LC MS workflow implemented on hybrid quadrupole time of flight ToF instrument to detect modified peptide in complex peptide sample and establish the nature of the modification the method is based on the alternating acquisition of full mass spectra under different collision conditions inducing the cleavage of the substituents modified peptide are detected based on their specific fragmentation generating the nonmodi fied peptide backbone and reporter ions in the low mass region the two mass analyzer stages of ToF instrument are used to eliminate the low mass chemical background in the quadrupole and thus facilitate the detection of low mass reporter ions in the ToF off line data processing enables detection of one or even multiple modifications and the modified candidates are subsequently sequenced in directed MS MS mode the technique was applied to the analysis of glcNAc pep tides very complex mixture of linked glycopeptides and phosphotyrosine peptide

although the classification of cell types often relies on the identification of cell surface protein as differentiation markers flow cytometry requires suitable antibodies and currently permits detection of only up to dozen differentiation markers in single measurement We use multiplexed mass spectrometric identification of several hundred linked glycosylation sites specifically from cell surface exposed glycoproteins to phenotype cell without antibodies in an unbiased fashion and without priori knowledge We apply our cell surface capturing CSC technology which covalently labels extracellular glycan moieties on live cell to the detection and relative quantitative comparison of the cell surface glycoproteomes of and cell as well as to monitor changes in the abundance of cell surface glycoprotein markers during cell activation and the controlled differentiation of embryonic stem cell into the neural lineage snapshot view of the cell surface glycoproteins will enable detection of panels of glycoproteins as potential differentiation markers that are currently not accessible by other means

We present mass spectrometry based strategy for the specific detection and quantification of cell surface proteome changes the method is based on the label free quantification of peptide patterns acquired by high mass accuracy mass spectrometry using new software tools and the cell surface capturing technology that selectively enriches glycopeptides exposed to the cell exterior the method was applied to monitor dynamic protein changes in the cell surface glycoproteome of drosophila melanogaster cell the results led to the construction of cell surface glycoprotein atlas consisting of cell surface glycoproteins of melanogaster Kc167 cell and indicated relative quantitative changes of cell surface glycoproteins in four different cellular states furthermore we specifically investigated cell surface proteome changes upon prolonged insulin stimulation the data revealed insulin dependent cell surface glycoprotein dynamics including insulin receptor internalization and linked these changes to intracellular signaling networks

ubiquitin like protein UBLs can change protein function localization or turnover by covalent attachment to lysine residue although UBLs achieve this conjugation through an intricate enzymatic cascade their bacterial counterparts moaD and thiS function as sulphur carrier protein here we show that urm1p the most ancient UBL acts as sulphur carrier in the process of eukaryotic transfer RNA tRNA modification providing possible evolutionary link between UBL and sulphur transfer moreover we identify uba4p ncs2p ncs6p and yor251cp as components of this conserved pathway using in vitro assay we show that ncs6p binds to tRNA whereas uba4p first adenylates and then directly transfers sulphur onto urm1p finally functional analysis reveals that the thiolation function of urm1p is critical to regulate cellular responses to nutrient starvation and oxidative stress conditions most likely by increasing translation fidelity macmillan publishers limited all rights reserved

We describe an integrative software platform prequips for comparative proteomics based system biology analysis that integrates all information generated from mass spectrometry MS based proteomics as well as from basic proteomics data analysis tools ii visualizes such information for various proteomic analyses via graphical interfaces and iii links peptide and protein abundances to external tools often used in system biology studies

the nematode caenorhabditis elegans is popular model system in genetics not least because majority of human disease gene are conserved in elegans To generate comprehensive inventory of its expressed proteome we performed extensive shotgun proteomics and identified more than half of all predicted elegans protein this allowed us to confirm and extend genome annotations characterize the role of operons in elegans and semiquantitatively infer abundance levels for thousands of protein furthermore for the first time to our knowledge we were able to compare two animal proteomes elegans and drosophila melanogaster We found that the abundances of orthologous protein in metazoans correlate remarkably well better than protein abundance versus transcript abundance within each organism or transcript abundances across organisms this suggests that changes in transcript abundance may have been partially offset during evolution by opposing changes in protein abundance copyright

identification of the protein constituents of cell organelles forms the basis for studies to define the roles of specific protein in organelle structure and functions over the past decade the use of mass spectrometry based proteomics has dissected various organelles and allowed the association of many novel protein with particular organelles this review chronicles the evolution of organelle proteomics technology and discusses how many limitations such as organelle heterogeneity and purity can be avoided with recently developed quantitative profiling approaches although many challenges remain quantitative profiling of organelles holds the promise to begin to address the complex and dynamic shuttling of protein among organelles that will be critical for application of this advanced technology to disease based changes in organelle function

background crucial foundations of any quantitative system biology experiment are correct genome and proteome annotations protein databases compiled from high quality empirical protein identifications that are in turn based on correct gene models increase the correctness sensitivity and quantitative accuracy of system biology genome scale experiments results In this manuscript we present the drosophila melanogaster peptideatlas fly proteomics and genomics resource of unsurpassed depth based on peptide mass spectrometry data collected in our laboratory the portal http www drosophila peptideatlas org allows querying fly protein data observed with respect to gene model confirmation and splice site verification as well as for the identification of proteotypic peptide suited for targeted proteomics studies additionally the database provides consensus mass spectra for observed peptide along with qualitative and quantitative information about the number of observations of particular peptide and the sample in which it was observed conclusion peptideatlas is an open access database for the drosophila community that has several features and applications that support reduction of the complexity inherently associated with performing targeted proteomic studies designing and accelerating shotgun proteomics experiments confirming or questioning gene models and adjusting gene models such that they are in line with observed drosophila peptide while the database consists of proteomic data it is not required that the user is proteomics expert

We present mass spectrometry based strategy for the absolute quantification of protein complex components isolated through affinity purification We quantified bait protein via isotope labeled reference peptide corresponding to an affinity tag sequence and prey protein by label free correlational quantification using the precursor ion signal intensities of proteotypic peptide generated in reciprocal purifications We used this method to quantitatively analyze interaction stoichiometries in the human protein phosphatase 2A network

the high complexity and large dynamic range of blood plasma protein currently prohibit the sensitive and high throughput profiling of disease and control plasma proteome sample sets large enough to reliably detect disease indicating differences To circumvent these technological limitations we describe here new two stage strategy for the mass spectrometry MS assisted discovery verification and validation of disease biomarkers In an initial discovery phase linked glycoproteins with distinguishable expression patterns in primary normal and diseased tissue are detected and identified In the second step the protein identified in the initial phase are subjected to targeted MS analysis in plasma samples using the highly sensitive and specific selected reaction monitoring SRM technology since glycosylated protein such as those secreted or shed from the cell surface are likely to reside and persist in blood the two stage strategy is focused on the quantification of tissue derived glycoproteins in plasma the focus on the glycoproteome not only reduces the complexity of the analytes but also targets an information rich subproteome which is relevant for remote sensing of diseases in the plasma the glycoprotein based biomarker discovery and validation workflow reviewed here allows for the robust identification of protein candidate panels that can finally be selectively monitored in the blood plasma at high sensitivity in reliable non invasive and quantitative fashion

current mass spectrometers provide number of alternative methodologies for producing tandem mass spectra specifically for phosphopeptide analysis In particular generation of MS spectra in data dependent manner upon detection of the neutral loss of phosphoric acid in MS spectra is popular technique for circumventing the problem of poor phosphopeptide backbone fragmentation the newer multistage activation method provides another option both these strategies require additional cycle time on the instrument and therefore reduce the number of spectra that can be measured in the same amount of time additional informatics is often required to make most efficient use of the additional information provided by these spectra as well this work presents comparison of several commonly used mass spectrometry method for the study of phosphopeptide enriched samples an MS only method multistage activation method and an MS MS data dependent neutral loss method several strategies for dealing effectively with the resulting MS data in the latter approach are also presented and compared the overall goal is to infer whether any one methodology performs significantly better than another for identifying phosphopeptides On data presented here the multistage activation methodology is demonstrated to perform optimally and does not result in significant loss of unique peptide identifications

the recent advance in technology for mass spectrometry based targeted protein quantification has opened new avenues for broad range of proteomic applications in clinical research the major breakthroughs are highlighted by the capability of using universal approach to perform quantitative assay for wide spectrum of protein with minimum restrictions and the ease of assembling multiplex detections in single measurement the quantitative approach relies on the use of synthetic stable isotope labeled peptide or protein which precisely mimic their endogenous counterparts and act as internal standards to quantify the corresponding candidate protein this report reviews recently developed platform technologies for emerging applications of clinical proteomics and biomarker development

the main conclusion is that system biology approaches can indeed advance cancer research having already proved successful in very wide variety of cancer related areas and are likely to prove superior to many current research strategies major points include system biology and computational approaches can make important contributions to research and development in key clinical aspects of cancer and of cancer treatment and should be developed for understanding and application to diagnosis biomarkers cancer progression drug development and treatment strategies development of new measurement technologies is central to successful system approaches and should be strongly encouraged the system view of disease combined with these new technologies and novel computational tools will over the next years lead to medicine that is predictive personalized preventive and participatory P4 medicine major initiatives are in progress to gather extremely wide ranges of data for both somatic and germ line genetic variations as well as gene transcript protein and metabolite expression profiles that are cancer relevant electronic databases and repositories play central role to store and analyze these data these resources need to be developed and sustained understanding cellular pathways is crucial in cancer research and these pathways need to be considered in the context of the progression of cancer at various stages At all stages of cancer progression major areas require modelling via system and developmental biology method including immune system reactions angiogenesis and tumour progression number of mathematical models of an analytical or computational nature have been developed that can give detailed insights into the dynamics of cancer relevant system these models should be further integrated across multiple levels of biological organization in conjunction with analysis of laboratory and clinical data biomarkers represent major tools in determining the presence of cancer its progression and the responses to treatments there is need for sets of high quality annotated clinical samples enabling comparisons across different diseases and the quantitative simulation of major pathways leading to biomarker development and analysis of drug effects education is recognized as key component in the success of any system biology programme especially for applications to cancer research It is recognized that balance needs to be found between the need to be interdisciplinary and the necessity of having extensive specialist knowledge in particular areas proposal from this workshop is to explore one or more types of cancer over the full scale of their progression for example glioblastoma or colon cancer such an exemplar project would require all the experimental and computational tools available for the generation and analysis of quantitative data over the entire hierarchy of biological information these tools and approaches could be mobilized to understand detect and treat cancerous process and establish method applicable across wide range of cancers

dysfunction and loss of insulin producing pancreatic cell represent hallmarks of diabetes mellitus here we show that mice lacking the mitogen activated protein kinase MAPK p38d display improved glucose tolerance due to enhanced insulin secretion from pancreatic cell deletion of p38d results in pronounced activation of protein kinase PKD the latter of which we have identified as pivotal regulator of stimulated insulin exocytosis p38d catalyzes an inhibitory phosphorylation of PKD1 thereby attenuating stimulated insulin secretion In addition p38d null mice are protected against high fat feeding induced insulin resistance and oxidative stress mediated cell failure inhibition of PKD1 reverses enhanced insulin secretion from p38d deficient islets and glucose tolerance in p38d null mice as well as their susceptibility to oxidative stress In conclusion the p38d PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic cell pointing to pivotal role for this pathway in the development of overt diabetes mellitus

protein complexes represent major functional units for the execution of biological process systematic affinity purification coupled with mass spectrometry AP MS yielded wealth of information on the compendium of protein complexes expressed in saccharomyces cerevisiae however global AP MS analysis of human protein complexes is hampered by the low throughput sensitivity and data robustness of existing procedures which limit its application for system biology research here we address these limitations by novel integrated method which we applied and benchmarked for the human protein phosphatase 2A system We identified total of protein interactions with high reproducibility showing the coexistence of distinct classes of phosphatase complexes that are linked to protein implicated in mitosis cell signalling DNA damage control and more these results show that the presented analytical process will substantially advance throughput and reproducibility in future systematic AP MS studies on human protein complexes

legionella pneumophila the causative agent of legionnaires disease replicates in macrophages and amoebae within legionella containing vacuoles LCVs which communicate with the early secretory pathway and the endoplasmic reticulum formation of LCVs requires the bacterial icm dot type IV secretion system the icm dot translocated effector protein sidC selectively anchors to LCVs by binding the host lipid phosphatidylinositol phosphate ptdins here we describe novel and simple approach to purify intact vacuoles formed by pneumophila within dictyostelium discoideum by using magnetic immunoseparation with an antibody against sidC followed by density gradient centrifugation To monitor LCV purification by fluorescence microscopy we used dictyostelium producing the LCV marker calnexin GFP and pneumophila labeled with the red fluorescent protein Dsred proteome analysis of purified LCVs by liquid chromatography coupled to tandem mass spectrometry revealed host protein including known LCV components such as the small GTpases arf1 rab1 and rab7 rab8 an endosomal regulator of the late secretory pathway originating from the trans golgi network and the endosomal GTpase rab14 were identified as novel LCV components which were found to be present on vacuoles harboring wild type but not icm dot deficient pneumophila thus LCVs also communicate with the late secretory and endosomal pathways depletion of rab8 or arf1 by RNA interference reduced the amount of sidC on LCVs indicating that the GTpases promote the recruitment of legionella effectors by regulating the level of ptdins

the serine threonine protein phosphatases are targeted to specific subcellular locations and substrates in part via interactions with wide variety of regulatory protein understanding these interactions is thus critical to understanding phosphatase function using an iterative affinity purification mass spectrometry approach we generated high density interaction map surrounding the protein phosphatase 2A catalytic subunit this approach recapitulated the assembly of the PP2A catalytic subunit into many different trimeric complexes but also revealed several new protein protein interactions here we define novel large multiprotein assembly referred to as the striatin interacting phosphatase and kinase STRIPAK complex STRIPAK contains the PP2A catalytic PP2Ac and scaffolding PP2A subunits the striatins PP2A regulatory subunits the striatin associated protein mob3 the novel protein STRIP1 and STRIP2 formerly FAM40A and FAM40B the cerebral cavernous malformation CCM3 protein and members of the germinal center kinase III family of ste20 kinases although the function of the CCM3 protein is unknown the CCM3 gene is mutated in familial cerebral cavernous malformations condition associated with seizures and strokes our proteomics survey indicates that large portion of the CCM3 protein resides within the STRIPAK complex opening the way for further studies of CCM3 biology the STRIPAK assembly establishes mutually exclusive interactions with either the CTTNBP2 protein which interact with the cytoskeletal protein cortactin or second subcomplex consisting of the sarcolemmal membrane associated protein SLMAP and the related coiled coil protein suppressor of IKKe SIKE and FGFR1OP2 We have thus identified several novel PP2A containing protein complexes including large assembly linking kinases and phosphatases to gene mutated in human disease

the HUPO plasma proteome project new phase PPP held its initial workshop on august at the 7th world congress of proteomics in amsterdam technology platforms data repositories informatics and engagement of research groups for the submission of major datasets were key topics plasma is expected to be the common pathway for biomarker development and application through collaboration and integration with other HUPO initiatives

background quantitative proteomics holds great promise for identifying protein that are differentially abundant between populations representing different physiological or disease states range of computational tools is now available for both isotopically labeled and label free liquid chromatography mass spectrometry LC MS based quantitative proteomics however they are generally not comparable to each other in terms of functionality user interfaces information input output and do not readily facilitate appropriate statistical data analysis these limitations along with the array of choices present daunting prospect for biologists and other researchers not trained in bioinformatics who wish to use LC MS based quantitative proteomics results We have developed corra computational framework and tools for discovery based LC MS proteomics corra extends and adapts existing algorithms used for LC MS based proteomics and statistical algorithms originally developed for microarray data analyses appropriate for LC MS data analysis corra also adapts software engineering technologies google web toolkit distributed processing so that computationally intense data processing and statistical analyses can run on remote server while the user controls and manages the process from their own computer via simple web interface corra also allows the user to output significantly differentially abundant LC MS detected peptide features in form compatible with subsequent sequence identification via tandem mass spectrometry MS MS We present two case studies to illustrate the application of corra to commonly performed LC MS based biological workflows pilot biomarker discovery study of glycoproteins isolated from human plasma samples relevant to type diabetes and study in yeast to identify in vivo targets of the protein kinase ark1 via phosphopeptide profiling conclusion the corra computational framework leverages computational innovation to enable biologists or other researchers to process analyze and visualize LC MS data with what would otherwise be complex and not user friendly suite of tools corra enables appropriate statistical analyses with controlled false discovery rates ultimately to inform subsequent targeted identification of differentially abundant peptide by MS MS for the user not trained in bioinformatics corra represents complete customizable free and open source computational platform enabling LC MS based proteomic workflows and as such addresses an unmet need in the LC MS proteomics field

organelle protein profiles have traditionally been analyzed by subcellular fractionation of specific organelle followed by the identification of the protein components of specific fractions containing the target organelle using mass spectrometry MS however because of limited resolution of the available fractionation method it is often difficult to isolate and thus profile pure organelles furthermore many protein secretory protein are often observed to dynamically shuttle between organelles therefore the determination of their true cellular localization requires the concurrent analysis of multiple organelles from the same cell lysate here we report an integrated experimental approach that simultaneously profiles multiple organelles It is based on the subcellular fractionation of cell lysates by density gradient centrifugation iTRAQ labeling and MS analysis of the protein in selected fractions and principal component analysis PCA of the resulting quantitative proteomic data quantitative signature patterns of several organelles including the ribosome mitochondria proteasome lysosome endoplasmic reticulum ER and golgi apparatus have been acquired from single multiplexed proteomic assay using iTRAQ reagents through comparison PCA we compare organelle profiles from cell under different physiological conditions to investigate changes in organelle profiles between control and perturbed samples such quantitative proteomics based subcellular profiling method thus provide useful tools to dissect the organization of cellular protein into functional units and to detect dynamic changes in their protein composition

introduction proof of concept demonstration of the use of label free quantitative glycoproteomics for biomarker discovery workflow is presented in this paper using mouse model for skin cancer as an example materials and method blood plasma was collected from ten control mice and ten mice having mutation in the p19ARF gene conferring them high propensity to develop skin cancer after carcinogen exposure We enriched for glycosylated plasma protein ultimately generating deglycosylated forms of the tryptic peptide for liquid chromatography mass spectrometry LC MS analyses LC MS runs for each sample were then performed with view to identifying protein that were differentially abundant between the two mouse populations We then used recently developed computational framework corra to perform peak picking and alignment and to compute the statistical significance of any observed changes in individual peptide abundances once determined the most discriminating peptide features were then fragmented and identified by tandem mass spectrometry with the use of inclusion lists results and discussions We assessed the identified protein to see if there were sets of protein indicative of specific biological process that correlate with the presence of disease and specifically cancer according to their functional annotations As expected for such sick animals many of the protein identified were related to host immune response however significant number of protein are also directly associated with process linked to cancer development including protein related to the cell cycle localization transport and cell death additional analysis of the same samples in profiling mode and in triplicate confirmed that replicate MS analysis of the same plasma sample generated less variation than that observed between plasma samples from different individuals demonstrating that the reproducibility of the LC MS platform was sufficient for this application conclusion these results thus show that an LC MS based workflow can be useful tool for the generation of candidate protein of interest as part of disease biomarker discovery effort

background alternative splicing of messenger RNA permits the formation of wide range of mature RNA transcripts and has the potential to generate diverse spectrum of functional protein although there is extensive evidence for large scale alternative splicing at the transcript level there have been no comparable studies demonstrating the existence of alternatively spliced protein isoforms results recent advances in proteomics technology have allowed us to carry out comprehensive identification of protein isoforms in drosophila the analysis of this proteomic data confirmed the presence of multiple alternative gene products for over hundred drosophila gene conclusions We demonstrate that proteomics techniques can detect the expression of stable alternative splice isoforms on genome wide scale many of these alternative isoforms are likely to have regions that are disordered in solution and specific proteomics methodologies may be required to identify these peptide

the 7th world congress of the human proteome organization HUPO was held in amsterdam the netherlands from august to the event offered very dense day agenda consisting of an exciting scientific program documenting the tremendous progress the current challenges and the major recent accomplishments of proteomics an exhibition in which all the major vendors in the field of proteornics from around the globe showcased their products technologies and services educational and training events in which new proteomic technologies were introduced and taught and series of workshops and discussion forums of all HUPO sanctioned initiatives including potential human proteome project around scientific abstracts were received companies signed up for and supported the exhibition and the total number of registrations was just above with close to also present for the weekend pre program more than nationalities were represented at the meeting

summary MS BID MS biomarker discovery platform is an integrative computational pipeline for biomarker discovery using LC MS based comparative proteomic analysis this platform consists of several computational tools for detecting peptide in the collected patterns ii matching detected peptide across number of LC MS datasets and iii selecting discriminatory peptide between classes of samples

LC MS MS has emerged as the method of choice for the identification and quantification of protein sample mixtures for very complex samples such as complete proteomes the most commonly used LC MS MS method data dependent acquisition DDA precursor selection is of limited utility the limited scan speed of current mass spectrometers along with the highly redundant selection of the most intense precursor ions generates bias in the pool of identified protein toward those of higher abundance directed LC MS MS approach that alleviates the limitations of DDA precursor ion selection by decoupling peak detection and sequencing of selected precursor ions is presented In the first stage of the strategy all detectable peptide ion signals are extracted from high resolution LC MS feature maps or aligned sets of feature maps the selected features or subset thereof are subsequently sequenced in sequential non redundant directed LC MS MS experiments and the MS MS data are mapped back to the original LC MS feature map in fully automated manner the strategy implemented on an LTQ FT MS platform allowed the specific sequencing of features per analysis and enabled the identification of more than phosphorylation sites using single reversed phase separation dimension without the need for time consuming prefractionation steps compared with conventional DDA LC MS MS experiments substantially higher number of peptide could be identified from sample and this increase was more pronounced for low intensity precursor ions

systems biology relies on data sets in which the same group of protein is consistently identified and precisely quantified across multiple samples requirement that is only partially achieved by current proteomics approaches selected reaction monitoring SRM also called multiple reaction monitoring is emerging as technology that ideally complements the discovery capabilities of shotgun strategies by its unique potential for reliable quantification of analytes of low abundance in complex mixtures In an SRM experiment predefined precursor ion and one of its fragments are selected by the two mass filters of triple quadrupole instrument and monitored over time for precise quantification series of transitions precursor fragment ion pairs in combination with the retention time of the targeted peptide can constitute definitive assay typically large number of peptide are quantified during single LC MS experiment this tutorial explains the application of SRM for quantitative proteomics including the selection of proteotypic peptide and the optimization and validation of transitions furthermore normalization and various factors affecting sensitivity and accuracy are discussed

background protein phosphorylation regulates multitude of biological process however the large number of protein kinases and their substrates generates an enormously complex phosphoproteome the cyclin dependent kinases the CDKs comprise class of enzymes that regulate cell cycle progression and play important roles in tumorigenesis however despite intense study only limited number of mammalian CDK substrates are known comprehensive understanding of CDK function requires the identification of their substrate network results We describe simple and efficient approach to identify potential cyclin CDK2 targets in complex cell lysates using kinase engineering strategy combined with chemical enrichment and mass spectrometry we identified potential cyclin CDK2 substrates and more than phosphorylation sites about of these candidates function within pathways related to cell division and the vast majority are involved in other fundamental cellular process We have validated several candidates as direct cyclin CDK2 substrates that are phosphorylated on the same sites that we identified by mass spectrometry and we also found that one novel substrate the ribosomal protein RL12 exhibits site specific CDK2 dependent phosphorylation in vivo conclusions We used method entailing engineered kinases and thiophosphate enrichment to identify large number of candidate CDK2 substrates in cell lysates these results are consistent with other recent proteomic studies and suggest that CDKs regulate cell division via large networks of cellular substrates these method are general and can be easily adapted to identify direct substrates of many other protein kinases

spectral searching has drawn increasing interest as an alternative to sequence database searching in proteomics We developed and validated an open source software toolkit spectraST to enable proteomics researchers to build spectral libraries and to integrate this promising approach in their data analysis pipeline It allows individual researchers to condense raw data into spectral libraries summarizing information about observed proteomes into concise and retrievable format for future data analyses

mass spectrometry experiments in the field of proteomics produce lists containing tens to thousands of identified protein with the protein information and property explorer PIPE the biologist can acquire functional annotations for these protein and explore the enrichment of the list or fraction thereof with respect to functional classes these protein lists may be saved for access at later time or different location the PIPE is interoperable with the firegoose and the gaggle permitting wide ranging data exploration and analysis the PIPE is rich client web application which uses AJAX capabilities provided by the google web toolkit and server side data storage using hibernate

In many studies particularly in the field of system biology it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states the high degree of reproducibility required for such experiments has not been achieved by classical mass spectrometry based proteomics method In this study we describe the implementation of targeted quantitative approach by which predetermined protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples this approach consists of three steps first the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry and the data generated are assembled in the peptideatlas database second based on this proteome map peptide uniquely identifying the protein of interest proteotypic peptide are selected and multiple reaction monitoring MRM transitions are established and validated by MS2 spectrum acquisition this process of peptide selection transition selection and validation is supported by suite of software tools TIQAM targeted identification for quantitative analysis by MRM described in this study third the selected target protein set is quantified in multiple samples by MRM applying this approach we were able to reliably quantify low abundance virulence factors from cultures of the human pathogen streptococcus pyogenes exposed to increasing amounts of plasma the resulting quantitative protein patterns enabled us to clearly define the subset of virulence protein that is regulated upon plasma exposure

cohesin is required to prevent premature dissociation of sister chromatids after DNA replication although its role in chromatid cohesion is well established the functional significance of cohesin association with interphase chromatin is not clear using quantitative proteomics approach we show that the STAG1 scc3 SA1 subunit of cohesin interacts with the CCTC binding factor CTCF bound to the myc insulator element both allele specific binding of CTCF and scc3 SA1 at the imprinted IGF2 H19 gene locus and our analyses of human DM1 alleles containing base substitutions at CTCF binding motifs indicate that cohesin recruitment to chromosomal sites depends on the presence of CTCF large scale genomic survey using ChIP chip demonstrates that scc3 SA1 binding strongly correlates with the CTCF binding site distribution in chromosomal arms however some chromosomal sites interact exclusively with CTCF whereas others interact with scc3 SA1 only furthermore immunofluorescence microscopy and ChIP chip experiments demonstrate that CTCF associates with both centromeres and chromosomal arms during metaphase these results link cohesin to gene regulatory functions and suggest an essential role for CTCF during sister chromatid cohesion these results have implications for the functional role of cohesin subunits in the pathogenesis of cornelia de lange syndrome and roberts syndromes

We present an in depth analysis of mouse plasma leading to the development of publicly available repository composed of liquid chromatography tandem mass spectrometry runs total of distinct peptide have been identified with high confidence the corresponding approximately protein are estimated to span logarithmic range of abundance in plasma major finding from this study is the identification of novel isoforms and transcript variants not previously predicted from genome analysis

A crucial part of successful system biology experiment is an assay that provides reliable quantitative measurements for each of the components in the system being studied for proteomics to be key part of such studies it must deliver accurate quantification of all the components in the system for each tested perturbation without any gaps in the data this will require new approach to proteomics that is based on emerging targeted quantitative mass spectrometry techniques the peptideatlas project comprises growing publicly accessible database of peptide identified in many tandem mass spectrometry proteomics studies and software tools that allow the building of peptideatlas as well as its use by the research community here we describe the peptideatlas project its contents and components and show how together they provide unique platform to select and validate mass spectrometry targets thereby allowing the next revolution in proteomics

BACKGROUND malignant pleural effusion of advanced lung adenocarcinoma may be valid source for detection of biomarkers such as glycosylated protein GP because tumor cell grow during weeks in this liquid the authors aimed for creation of GP effusion profiles from routine cytology specimens to detect relevant biomarkers method hundred microliters of malignant pleural effusions of patients with lung adenocarcinoma and nonmalignant controls were used for triplicate GP capture by solid phase extraction after trypsin digest and PNgase release liquid chromatography separation connected online to tandem mass spectrometer was performed by liquid chromatography tandem mass spectrometry LC MS MS RESULTS In the total of samples and nonredundant protein were detected with probabilities of and respectively the specificity for the glycomotif was at penetration into the moderate to low protein concentration range µg ng mL occurred and several protein associated with tumor progression or metastasis were identified including CA CD44 CD166 lysosome associated membrane glycoprotein LAMP multimerin and periostin MS identifications were correlated with the corresponding immunoreactivity in either effusion fluid or tumor tissue CONCLUSIONS In conclusion reduction of sample complexity by GP capturing allows detection of protein in the µg to ng mL range pleural effusion is useful source for biomarker research in lung cancer

We describe method to identify cross linked peptide from complex samples and large protein sequence databases by combining isotopically tagged cross linkers chromatographic enrichment targeted proteomics and new search engine called xquest this software reduces the search space by an upstream candidate peptide search before the recombination step We showed that xquest can identify cross linked peptide from total escherichia coli lysate with an unrestricted database search

absolute quantification of peptide by mass spectrometry requires reference frequently using heavy isotope coded peptide as internal standards these peptide have traditionally been generated by chemical stepwise synthesis recently new way to supply such peptide was described in which nucleotide sequence coding for the respective peptide are concatenated into synthetic gene qconCAT these qconCATs are then expressed to produce polypeptide consisting of concatenated peptide purified quantified by various method and then digested to yield the final internal standard peptide although both of these method for peptide production are routinely used for absolute quantifications there is currently no information regarding the accuracy of the quantifications made in each case In this study we used sets of synthetic and biological peptide in parallel to evaluate the accuracy of either method We also addressed some technical issues regarding the preparation and proper utilization of such standard peptide twenty five peptide derived from the caenorhabditis elegans proteome were selected for this study twenty four were successfully chemically synthesized five oconCAT gene were designed each concatenation of the same peptide but each in separate different randomized order and expressed via in vitro translation reactions that contained heavy isotope labeled lysine and arginine three of the five qconCATs were successfully produced different digestion conditions including various detergents and incubation conditions were tested to find those optimal for the generation of reproducible and accurate reference sample mixture all three qconCAT polypeptides were then digested using the optimized conditions and then mixed in ratio with their synthetic counterparts multireaction monitoring mass spectrometry was then used for quantification results showed that the digestion protocol had significant impact on equimolarity of final peptide confirming the need for optimization under optimal conditions however most qconCAT peptide were produced at equimolar ratio few qconCAT derived peptide were largely overestimated due to problems with solubilization or stability of the synthetic peptide although the order in which the peptide sequence appeared in the qconCAT sequence proved to affect the success rate of in vitro translation it did not significantly affect the final peptide yields overall neither the chemical synthesis nor recombinant genetic approach proved to be superior method for the production of reference peptide for absolute quantification

data processing is central and critical component of successful proteomics experiment and is often the most time consuming step there have been considerable advances in the field of proteomics informatics in the past years spurred mainly by free and open source software tools along with the gains afforded by new software the benefits of making raw data and processed results freely available to the community in data repositories are finally in evidence In this review we provide an overview of the general analysis approaches software tools and repositories that are enabling successful proteomics research via tandem mass spectrometry copyright

enhancers have been functionally described for years but the molecular principles underlying the integration of regulatory inputs to alternate gene enhancers used during mammalian organogenesis remain incompletely understood using combination of in vivo enhancer mapping and proteomics approaches we have established that two distant and distinct early enhancers each requiring different transcription complexes are required for full activation of the gene encoding the pituitary lineage determining factor pit1 transcription factor belonging to the giant multiple homeodomain and zinc finger family atbf1 serves as novel pituitary regulator for one of the two required enhancers as shown by genetic and in vitro analysis

solid phase extraction of glycopeptides SPEG coupled with quantitative proteomic analysis using mass spectrometry has shown great potential for investigating glycoproteins in an effort to discover new diagnostic biomarkers or therapeutic targets As solid phase approach SPEG can be performed with microtiter plate to provide high throughput platform for large scale screening of clinical samples here we describe the synthesis of superparamagnetic silica particles with hydrazide groups on the surface and further evaluate their use as the solid support for SPEG We produced nonspherical silica particles containing superparamagnetic iron oxide cores using modified Stöber method and then derivatized their surface with hydrazide terminated silane such composite particles displayed strong response to the external magnetic field and this feature enabled us to capture and release the particles easily for automated high throughput sample preparation of glycopeptides when measured with standard glycoproteins the adsorption capacity of these particles was mg of glycoproteins per of nanoparticles the nanoparticles were used in microtiter plate format for glycopeptide capture using liquid handler the captured glycopeptides were then analyzed by LC MS and LC MS MS to determine the specificity and reproducibility of glycopeptide isolation our results demonstrate the potential of these superparamagnetic colloidal particles for high throughput analysis of glycoproteins

protein complexes have largely been studied by immuno affinity purification and mass spectrometric analysis although this approach has been widely and successfully used it is limited because it has difficulties reliably discriminating true from false protein complex components identifying post translational modifications and detecting quantitative changes in complex composition or state of modification of complex components We have developed protocol that enables us to determine in single LC MALDI TOF TOF analysis the true protein constituents of complex to detect changes in the complex composition and to localize phosphorylation sites and estimate their respective stoichiometry the method is based on the combination of fourplex iTRAQ isobaric tags for relative and absolute quantification isobaric labeling and protein phosphatase treatment of substrates It was evaluated on model peptide and protein and on the complex ccl1 kin28 tfb3 isolated by tandem affinity purification from yeast cell the two known phosphosites in kin28 and tfb3 could be reproducibly shown to be fully modified the protocol was then applied to the analysis of samples immunopurified from drosophila melanogaster cell expressing an epitope tagged form of the insulin receptor substrate homologue chico these experiments allowed us to identify 3e and the insulin receptor as specific chico interactors In further experiment we compared the immunopurified materials obtained from tagged chico expressing cell that were either treated with insulin or left unstimulated this analysis showed that hormone stimulation increases the association of protein with chico and modulates several phosphorylation sites of the bait some of which are located within predicted recognition motives of protein

targeted mass spectrometry has the potential to generate the complete quantitative proteomic datasets required for system biology

over the past decade series of experimental strategies for mass spectrometry based quantitative proteomics and corresponding computational methodology for the processing of the resulting data have been generated We provide here an overview of the main quantification principles and available software solutions for the analysis of data generated by liquid chromatography coupled to mass spectrometry LC MS three conceptually different method to perform quantitative LC MS experiments have been introduced In the first quantification is achieved by spectral counting in the second via differential stable isotopic labeling and in the third by using the ion current in label free LC MS measurements We discuss here advantages and challenges of each quantification approach and assess available software solutions with respect to their instrument compatibility and processing functionality this review therefore serves as starting point for researchers to choose an appropriate software solution for quantitative proteomic experiments based on their experimental and analytical requirements

improvements in ion trap instrumentation have made dimensional mass spectrometry more practical the overall goal of the study was to describe model for making use of MS2 and MS3 information in mass spectrometry experiments We present statistical model for adjusting peptide identification probabilities based on the combined information obtained by coupling peptide assignments of consecutive MS2 and MS3 spectra using two data sets mixture of known protein and complex phosphopeptide enriched sample we demonstrate an increase in discriminating power of the adjusted probabilities compared with models using MS2 or MS3 data only this work also addresses the overall value of generating MS3 data as compared with an MS2 only approach with focus on the analysis of phosphopeptide data

tandem mass spectrometry MS MS is frequently used in the identification of peptide and protein typical proteomic experiments rely on algorithms such as SEQUEST and MASCOT to compare thousands of tandem mass spectra against the theoretical fragment ion spectra of peptide in database the probabilities that these spectrum to sequence assignments are correct can be determined by statistical software such as peptideprophet or through estimations based on reverse or decoy databases however many of the software applications that assign probabilities for MS MS spectra to sequence matches were developed using training data sets from 3D ion trap mass spectrometers given the variety of types of mass spectrometers that have become commercially available over the last years we sought to generate data set of reference data covering multiple instrumentation platforms to facilitate both the refinement of existing computational approaches and the development of novel software tools We analyzed the proteolytic peptide in mixture of tryptic digests of protein named the ISB standard protein mix using different mass spectrometers these include linear and 3D ion traps two quadrupole time of flight platforms qq TOF and two MALDI TOF TOF platforms the resulting data set which has been named the standard protein mix database consists of over million spectra in 150+ replicate runs on the mass spectrometers the data were inspected for quality of separation and searched using SEQUEST all data including the native raw instrument and mzXML formats and the peptideprophet validated peptide assignments are available at http regis web systemsbiology net publicdatasets

proteomic analyses are critically important for system biology because important aspects related to the structure function and control of biological system are only amenable by direct protein measurements It has become apparent that the current proteomics technologies are unlikely to allow routine quantitative measurements of whole proteomes We have therefore suggested and largely implemented two step strategy for quantitative proteome analysis In first step the discovery phase the proteome observable by mass spectrometry is extensively analyzed the resulting proteome catalog can then be used to select peptide specific to only one protein so called proteotypic peptide PTPs It represents the basis to realize sensitive robust and reproducible measurements based on targeted mass spectrometry of these PTPs in subsequent scoring phase In this extra view we describe the need for such proteome catalogs and their multiple benefits for catalyzing the shift towards targeted quantitative proteomic analysis and beyond We use the insulin signaling cascade as representative example to illustrate the limitations of currently used proteomics approaches for the specific analysis of individual pathway components and describe how the recently published drosophila proteome catalog already helped to overcome many of these limitations

pancreatic cancer is the fourth leading cause of cancer death in the US only could survive years after diagnosis biomarkers are desperately needed to improve earlier more curable cancer diagnosis and to develop new effective therapeutic targets the development of quantitative proteomics technologies in recent years offers great promise for understanding the complex molecular events of tumorigenesis at the protein level and has stimulated great interest in applying the technology for pancreatic cancer studies proteomic studies of pancreatic tissues juice serum plasma and cell lines have recently attempted to identify differentially expressed protein in pancreatic cancer to dissect the abnormal signaling pathways underlying oncogenesis and to detect new biomarkers It can be expected that the continuing evolution of proteomics technology with better resolution and sensitivity will greatly enhance our capability in combating pancreatic cancer

We present method to grid enable tandem mass spectrometry protein identification the implemented parallelization strategy embeds the open source tandem tool in grid enabled workflow this allows rapid analysis of large scale mass spectrometry experiments on existing heterogeneous hardware We have explored different data splitting schemes considering both splitting spectra datasets and protein databases and examine the impact of the different schemes on scoring and computation time while resulting peptide values exhibit fluctuation we show that these variations are small caused by statistical rather than numerical instability and are not specific to the grid environment the correlation coefficient of results obtained on standalone machine versus the grid environment is found to be better than for spectra and for protein identification demonstrating the validity of our approach finally we examine the effect of different splitting schemes of spectra and protein data on CPU time and overall wall clock time revealing that judicious splitting of both data sets yields best overall performance

the ability to analyze and understand the mechanisms by which cell process information is key question of system biology research such mechanisms critically depend on reversible phosphorylation of cellular protein process that is catalyzed by protein kinases and phosphatases here we present phosphopep database containing more than unique high confidence phosphorylation sites mapping to nearly gene models and distinct phosphoproteins of the drosophila melanogaster Kc167 cell line this constitutes the most comprehensive phosphorylation map of any single source to date To enhance the utility of phosphopep we also provide an array of software tools that allow users to browse through phosphorylation sites on single protein or pathways to easily integrate the data with other external data types such as protein protein interactions and to search the database via spectral matching finally all data can be readily exported for example for targeted proteomics approaches and the data thus generated can be again validated using phosphopep supporting iterative cycles of experimentation and analysis that are typical for system biology research

S6 kinase S6K1 acts to integrate nutrient and growth factor signals to promote cell growth but also cell survival as mitochondria tethered protein kinase that phosphorylates and inactivates the proapoptotic molecule BAD here we report that the prefoldin chaperone URI represents mitochondrial substrate of S6K1 In growth factor deprived or rapamycin treated cell URI forms stable complexes with protein phosphatase PP at mitochondria thereby inhibiting the activity of the bound enzyme growth factor stimulation induces disassembly of URI PP1 complexes through S6K1 mediated phosphorylation of URI at serine this activates PP1 dependent negative feedback program that decreases S6K1 activity and BAD phosphorylation thereby altering the threshold for apoptosis these findings establish URI and PP1 as integral components of an S6K1 regulated mitochondrial pathway dedicated in part to oppose sustained S6K1 survival signaling and to ensure that the mitochondrial threshold for apoptosis is set in accord with nutrient and growth factor availability

In this article we provide direct evidence that the evolutionarily conserved transcription elongation factor TFIIS functions during preinitiation complex assembly first we identified TFIIS in mass spectrometric screen of RNA polymerase II pol II preinitiation complexes PICs second we show that the association of TFIIS with promoter depends on functional PIC components including mediator and the SAGA complex third we demonstrate that TFIIS is required for efficient formation of active PICs using truncation mutants of TFIIS we find that the pol II binding domain is the minimal domain necessary to stimulate PIC assembly however efficient formation of active PICs requires both the pol II binding domain and the poorly understood terminal domain importantly domain III which is required for the elongation function of TFIIS is dispensable during PIC assembly the results demonstrate that TFIIS is PIC component that is required for efficient formation and or stability of the complex

the functional genomics experiment data model FuGE has been developed to facilitate convergence of data standards for high throughput comprehensive analyses in biology FuGE models the components of an experimental activity that are common across different technologies including protocols samples and data FuGE provides foundation for describing entire laboratory workflows and for the development of new data formats the microarray gene expression data society and the proteomics standards initiative have committed to using FuGE as the basis for defining their respective standards and other standards groups including the metabolomics standards initiative are evaluating FuGE in their development efforts adoption of FuGE by multiple standards bodies will enable uniform reporting of common parts of functional genomics workflows simplify data integration efforts and ease the burden on researchers seeking to fulfill multiple minimum reporting requirements such advances are important for transparent data management and mining in functional genomics and system biology

label free quantification of high mass resolution LC MS data has emerged as promising technology for proteome analysis computational method are required for the accurate extraction of peptide signals from LC MS data and the tracking of these features across the measurements of different samples We present here an open source software tool superhirn that comprises set of modules to process LC MS data acquired on high resolution mass spectrometer the program includes newly developed functionalities to analyze LC MS data such as feature extraction and quantification LC MS similarity analysis LC MS alignment of multiple datasets and intensity normalization these program routines extract profiles of measured features and comprise tools for clustering and classification analysis of the profiles superhirn was applied in an MS1 based profiling approach to benchmark LC MS dataset of complex protein mixtures with defined concentration changes We show that the program automatically detects profiling trends in an unsupervised manner and is able to associate protein to their correct theoretical dilution profile

the analysis of the large amount of data generated in mass spectrometry based proteomics experiments represents significant challenge and is currently bottleneck in many proteomics projects In this review we discuss critical issues related to data processing and analysis in proteomics and describe available method and tools We place special emphasis on the elaboration of results that are supported by sound statistical arguments

the detection and quantification of plasma serum protein at or below the ng ml concentration range are of critical importance for the discovery and evaluation of new protein biomarkers this has been achieved either by the development of high sensitivity ELISA or other immunoassays for specific protein or by the extensive fractionation of the plasma proteome followed by the mass spectrometric analysis of the resulting fractions the first approach is limited by the high cost and time investment for assay development and the requirement of validated target the second although reasonably comprehensive and unbiased is limited by sample throughput here we describe method for the detection of plasma protein at concentrations in the ng ml or sub ng ml range and their accurate quantification over orders of magnitude the method is based on the selective isolation of glycosites from the plasma proteome and the detection and quantification of targeted peptide in quadrupole linear ion trap instrument operated in the multiple reaction monitoring MRM mode the unprecedented sensitivity of the mass spectrometric analysis of minimally fractionated plasma samples is the result of the significantly reduced sample complexity of the isolated glycosites compared with whole plasma proteome digests and the selectivity of the MRM process precise quantification was achieved via stable isotope dilution by adding 13C and or 15N labeled reference analytes We also demonstrate the possibility of significantly expanding the number of MRM measurements during one single LC MS run without compromising sensitivity by including elution time constraints for the targeted transitions thus allowing quantification of large sets of peptide in single analysis

We describe the structure and function of the toposome modified calcium binding iron less transferrin the first member of new class of cell adhesion protein In addition to the amino-acid sequence of the precursor we determined by edman degradation the terminal amino-acid sequence of the mature hexameric glycoprotein present in the egg as well as that of its derived proteolytically modified fragments necessary for development beyond the blastula stage the approximate termini of the fragments were determined by combination of mass spectrometry and migration in reducing gels before and after deglycosylation this new member of the transferrin family shows special features which explain its evolutionary adaptation to development and adhesive function in sea urchin embryos protease inhibiting WAP domain ii amino-acid cysteine less insertion in the terminal lobe and iii residue terminal extension with modified cystine knot motif found in multisubunit external cell surface glycoproteins proteolytic removal of the terminal WAP domain generates the mature toposome present in the oocyte the modified cystine knot motif stabilizes cell bound trimers upon Ca dependent dissociation of hexamer linked cell We determined the positions of the developmentally regulated cuts in the cysteine less insertion which produce the fragments observed previously these fragments remain bound to the hexameric 22S particle in vivo and are released only after treatment of the purified toposome with reducing agents In addition some soluble smaller fragments with possible signal function are produced sequence comparison of five sea urchin species reveals the location of the cell cell contact site targeted by the species specific embryo dissociating antibodies the evolutionary tree of and ferric transferrins implies their evolution from basic cation activated allosteric design modified to serve multiple functions

mass spectrometry based proteomics holds great promise as discovery tool for biomarker candidates in the early detection of diseases recently much emphasis has been placed upon producing highly reliable data for quantitative profiling for which highly reproducible methodologies are indispensable the main problems that affect experimental reproducibility stem from variations introduced by sample collection preparation and storage protocols and LC MS settings and conditions On the basis of formally precise and quantitative definition of similarity between LC MS experiments we have developed cha order fully automatic software tool that can assess experimental reproducibility of sets of large scale LC MS experiments By visualizing the similarity relationships within set of experiments this tool can form the basis of systematic quality control and thus help assess the comparability of mass spectrometry data over time across different laboratories and between instruments applying chaorder to data from multiple laboratories and range of instruments experimental protocols and sample complexities revealed biases introduced by the sample processing steps experimental protocols and instrument choices moreover we show that reducing bias by correcting for just few steps for example randomizing the run order does not provide much gain in statistical power for biomarker discovery

the analysis by liquid chromatography coupled to tandem mass spectrometry of complex peptide mixtures generated by proteolysis of protein samples is the main proteomics method used today the approach is based on the assumption that each protein present in sample reproducibly and predictably generates relatively small number of peptide that can be identified by mass spectrometry In this study this assumption was examined by targeted peptide sequencing strategy using inclusion lists to trigger peptide fragmentation attempts It was found that the number of peptide observed from single protein is at least one order of magnitude greater than previously assumed this unexpected complexity of proteomics samples implies substantial technical challenges explains some perplexing results in the proteomics literature and prompts the need for developing alternative experimental strategies for the rapid and comprehensive analysis of proteomes

MLL containing complexes methylate histone H3 at lysine H3K4 and have been implicated in the regulation of transcription however it is unclear how MLL complexes are targeted to specific gene loci here we show that the MLL2 complex associates with the hematopoietic activator NF E2 in erythroid cell and is important for H3K4 trimethylation and maximal levels of transcription at the globin locus furthermore recruitment of the MLL2 complex to the globin locus is dependent upon NF E2 and coincides spatio temporally with NF E2 binding during erythroid differentiation thus DNA bound activator is important initially for guiding MLL2 to particular genomic location interestingly while the MLL2 associated subunit ASH2L is restricted to the globin locus control region kb upstream of the ßmaj globin gene the MLL2 protein spreads across the globin locus suggesting previously undefined mechanism by which an activator influences transcription and H3K4 trimethylation at distance

proteomic analysis of blood plasma can potentially identify biomarkers that are useful for classifying the physiological or pathological status of an individual and for monitoring the effects of therapy however the complexity of the plasma proteome the large number of peptide generated per protein due to dynamic protein post translational modifications of each protein and sequence variations among individuals pose great challenges to current proteomic technologies To overcome these challenges we have recently developed method for the high throughput analysis of glycoproteins using solid phase extraction of linked glycopeptides SPEG here we describe procedure for plasma analysis using SPEG in which each step of SPEG was optimized the performance of optimization was monitored using mouse plasma spiked with radioactive labeled human plasma glycoproteins our data show that standard procedure for plasma proteome analysis can be developed using the SPEG technique mainly due to the relatively constant protein content in plasma

the effective treatment of pancreatic cancer relies on the diagnosis of the disease at an early stage difficult challenge one major obstacle in the development of diagnostic biomarkers of early pancreatic cancer has been the dual expression of potential biomarkers in both chronic pancreatitis and cancer To better understand the limitations of potential protein biomarkers we used ICAT technology and tandem mass spectrometry based proteomics to systematically study protein expression in chronic pancreatitis among the differentially expressed protein identified in chronic pancreatitis most biological process were responses to wounding and inflammation finding consistent with the underlining inflammation and tissue repair associated with chronic pancreatitis furthermore of the differentially expressed protein identified in chronic pancreatitis have been implicated previously in pancreatic cancer suggesting some commonality in protein expression between these two diseases biological network analysis further identified MYC as common prominent regulatory protein in pancreatic cancer and chronic pancreatitis lastly five protein were selected for validation by western blot and immunohistochemistry annexin A2 and insulin like growth factor binding protein were overexpressed in cancer but not in chronic pancreatitis making them promising biomarker candidates for pancreatic cancer In addition our study validated that cathepsin integrin ß1 and plasminogen were overexpressed in both pancreatic cancer and chronic pancreatitis the positive involvement of these protein in chronic pancreatitis and pancreatic cancer will potentially lower the specificity of these protein as biomarker candidates for pancreatic cancer altogether our study provides some insights into the molecular events in chronic pancreatitis that may lead to diverse strategies for diagnosis and treatment of these diseases

both the generation and the analysis of proteomics data are now widespread and high throughput approaches are commonplace protocols continue to increase in complexity as method and technologies evolve and diversify To encourage the standardized collection integration storage and dissemination of proteomics data the human proteome organization proteomics standards initiative develops guidance modules for reporting the use of techniques such as gel electrophoresis and mass spectrometry this paper describes the process and principles underpinning the development of these modules discusses the ramifications for various interest groups such as experimentalists funders publishers and the private sector addresses the issue of overlap with other reporting guidelines and highlights the criticality of appropriate tools and resources in enabling MIAPE compliant reporting

the versatile combination of affinity purification and mass spectrometry AP MS has recently been applied to the detailed characterization of many protein complexes and large protein interaction networks the combination of AP MS with other techniques such as biochemical fractionation intact mass measurement and chemical crosslinking can help to decipher the supramolecular organization of protein complexes AP MS can also be combined with quantitative proteomics approaches to better understand the dynamics of protein complex assembly

mass spectrometry specifically the analysis of complex peptide mixtures by liquid chromatography and tandem mass spectrometry shotgun proteomics has been at the centre of proteomics research for the past decade To overcome some of the fundamental limitations of the approach including its limited sensitivity and high degree of redundancy new proteomic workflows are being developed among these targeting method in which specific peptide are selectively isolated identified and quantified are particularly promising here we summarize recent incremental advances in shotgun proteomic method and outline emerging targeted workflows the development of the target driven approaches with their ability to detect and quantify identical non redundant sets of protein in multiple repeat analyses will be crucially important for the application of proteomics to biomarker discovery and validation and to system biology research

mammalian neural stem cell NSCs have the capacity to both self renew and to generate all the neuronal and glial cell types of the adult nervous system global chromatin changes accompany the transition from proliferating NSCs to committed neuronal lineages but the mechanisms involved have been unclear using proteomics approach we show that switch in subunit composition of neural ATP dependent SWI SNF like chromatin remodeling complexes accompanies this developmental transition proliferating neural stem and progenitor cell express complexes in which BAF45a a Krüppel PHD domain protein and the actin related protein BAF53a are quantitatively associated with the SWI2 SNF2 like ATpases brg and brm As neural progenitors exit the cell cycle these subunits are replaced by the homologous BAF45b BAF45c and BAF53b BAF45a 53a subunits are necessary and sufficient for neural progenitor proliferation preventing the subunit switch impairs neuronal differentiation indicating that this molecular event is essential for the transition from neural stem progenitors to postmitotic neurons more broadly these studies suggest that SWI SNF like complexes in vertebrates achieve biological specificity by combinatorial assembly of their subunits

the goal of quantitative proteomics is to systematically study static state or perturbation induced changes in protein profile most of the recently developed mass spectrometry MS based quantitative proteomic method employ stable isotope labeling to introduce signature mass tags to peptide protein that can be used by mass spectrometer to quantify each analyte and to determine the sample from which it originates In this chapter we discuss several method for the introduction of mass tags to protein and peptide for MS based quantitative proteomic analysis including isotope coded affinity tags stable isotope labeling by amino-acid in cell culture global internal standard technology and mass coded abundance tagging

olfactory ensheathing cell OECs transplanted into the lesioned CNS can stimulate reportedly different degrees of regeneration remyelination and functional recovery but little is known about the mechanisms OECs may use to stimulate endogenous repair here we used functional proteomic approach isotope coded affinity tagging and mass spectrometry to identify active components of the OEC secreteome that differentially stimulate outgrowth SPARC secreted protein acidic rich in cysteine osteonectin was identified as an OEC derived matricellular protein that can indirectly enhance the ability of schwann cell to stimulate dorsal root ganglion outgrowth in vitro SPARC stimulates schwann cell mediated outgrowth by cooperative signal with laminin and transforming growth factor furthermore when SPARC null OECs were transplanted into lesioned rat spinal cord the absence of OEC secreted SPARC results in an attenuation of outgrowth of specific subsets of sensory and supraspinal axons and changes the pattern of macrophage infiltration in response to the transplanted cell these data provide the first evidence for role for SPARC in modulating different aspects of CNS repair and indicate that SPARC can change the activation state of endogenous schwann cell resulting in the promotion of outgrowth in vitro and in vivo copyright

understanding how protein and their complex interaction networks convert the genomic information into dynamic living organism is fundamental challenge in biological sciences As an important step towards understanding the system biology of complex eukaryote we cataloged of the predicted drosophila melanogaster proteome by detecting protein from redundant and distinct peptide identifications this unprecedented high proteome coverage for complex eukaryote was achieved by combining sample diversity multidimensional biochemical fractionation and analysis driven experimentation feedback loops whereby data collection is guided by statistical analysis of prior data We show that high quality proteomics data provide crucial information to amend genome annotation and to confirm many predicted gene models We also present experimentally identified proteotypic peptide matching of melanogaster gene models this library of proteotypic peptide should enable fast targeted and quantitative proteomic studies to elucidate the system biology of this model organism

integrated liquid chromatography mass spectrometry LC MS is becoming widely used approach for quantifying the protein composition of complex samples the output of the LC MS system measures the intensity of peptide with specific mass charge ratio and retention time In the last few years this technology has been used to compare complex biological samples across multiple conditions one challenge for comparative proteomic profiling with LC MS is to match corresponding peptide features from different experiments In this paper we propose new method peptide element alignment PETAL that uses raw spectrum data and detected peak to simultaneously align features from multiple LC MS experiments PETAL creates spectrum elements each of which represents the mass spectrum of single peptide in single scan peptide detected in different LC MS data are aligned if they can be represented by the same elements By considering each peptide separately PETAL enjoys greater flexibility than time warping method while most existing method process multiple data sets by sequentially aligning each data set to an arbitrarily chosen template data set PETAL treats all experiments symmetrically and can analyze all experiments simultaneously We illustrate the performance of PETAL on example data sets

current method for phosphoproteome analysis have several limitations first most method for phosphopeptide enrichment lack the specificity to truly purify phosphopeptides second fragmentation spectra of phosphopeptides in particular those of phosphoserine and phosphothreonine containing peptide are often dominated by the loss of the phosphate group and therefore lack the information required to identify the peptide sequence and the site of phosphorylation and third sequence database search engines and statistical models for data validation are not optimized for the specific fragmentation properties of phosphorylated peptide consequently phosphoproteomic data are characterized by large and unknown rates of false positive and false negative phosphorylation sites here we present an integrated chemical mass spectrometric and computational strategy to improve the efficiency specificity and confidence in the identification of phosphopeptides and their site of phosphorylation phosphopeptides were isolated with high specificity through simple derivatization procedure based on phosphoramidate chemistry identification of phosphopeptides their site of phosphorylation and the corresponding phosphoproteins was achieved by the optimization of the mass spectrometric data acquisition procedure the computational tools for database searching and the data post processing the strategy was applied to the mapping of phosphorylation sites of purified transcription factor dFOXO and for the global analysis of protein phosphorylation of drosophila melanogaster Kc167 cell this journal is

the ability to routinely analyze and quantitatively measure changes in protein phosphorylation on proteome wide scale is essential for biological and clinical research We assessed the ability of three common phosphopeptide isolation method phosphoramidate chemistry PAC immobilized metal affinity chromatography IMAC and titanium dioxide to reproducibly specifically and comprehensively isolate phosphopeptides from complex mixtures phosphopeptides were isolated from aliquots of tryptic digest of the cytosolic fraction of drosophila melanogaster Kc167 cell and analyzed by liquid chromatography electrospray ionization tandem mass spectrometry each method reproducibly isolated phosphopeptides the method however differed in their specificity of isolation and notably in the set of phosphopeptides isolated the results suggest that the three method detect different partially overlapping segments of the phosphoproteome and that at present no single method is sufficient for comprehensive phosphoproteome analysis

biological system are controlled by protein complexes that associate into dynamic protein interaction networks We describe strategy that analyzes protein complexes through the integration of label free quantitative mass spectrometry and computational analysis By evaluating peptide intensity profiles throughout the sequential dilution of samples the mastermap system identifies specific interaction partners detects changes in the composition of protein complexes and reveals variations in the phosphorylation states of components of protein complexes We use the complexes containing the human forkhead transcription factor foxO3A to demonstrate the validity and performance of this technology our analysis identifies previously known and unknown interactions of foxO3A with protein in addition to identifying foxO3A phosphorylation sites and detecting reduced binding following inhibition of phosphoinositide kinase By improving specificity and sensitivity of interaction networks assessing post translational modifications and providing dynamic interaction profiles the mastermap system addresses several limitations of current approaches for protein complexes

protein glycosylation is common post translational modification and has been increasingly recognized as one of the most prominent biochemical alterations associated with malignant transformation and tumorigenesis linked glycosylation is prevalent in protein on the extracellular membrane and many clinical biomarkers and therapeutic targets are glycoproteins here we describe protocol for solid phase extraction of linked glycopeptides and subsequent identification of linked glycosylation sites glycosites by tandem mass spectrometry the method oxidizes the carbohydrates in glycopeptides into aldehydes which can be immobilized on solid support the linked glycopeptides are then optionally labeled with stable isotope using deuterium labeled succinic anhydride and the peptide moieties are released by peptide glycosidase In single analysis the method identifies hundreds of linked glycoproteins the site of linked glycosylation and the relative quantity of the identified glycopeptides

A notable inefficiency of shotgun proteomics experiments is the repeated rediscovery of the same identifiable peptide by sequence database searching method which often are time consuming and error prone more precise and efficient method in which previously observed and identified peptide MS MS spectra are catalogued and condensed into searchable spectral libraries to allow new identifications by spectral matching is seen as promising alternative To that end an open source functionally complete high throughput and readily extensible MS MS spectral searching tool spectraST was developed high quality spectral library was constructed by combining the high confidence identifications of millions of spectra taken from various data repositories and searched using four sequence search engines the resulting library consists of over spectra for saccharomyces cerevisiae using this library spectraST vastly outperforms the sequence search engine SEQUEST in terms of speed and the ability to discriminate good and bad hits unique advantage of spectraST is its full integration into the popular trans proteomic pipeline suite of software which facilitates user adoption and provides important functionalities such as peptide and protein probability assignment quantification and data visualization this method of spectral library searching is especially suited for targeted proteomics applications offering superior performance to traditional sequence searching

there is intense interest in determining the absolute abundance of specific protein in complex mixtures for example in the area of disease biomarker discovery We have developed set of protein tagging reagents called visible isotope coded affinity tags VICAT reagents that contain protein tagging reagent for reaction with cysteine SH groups visible probe for monitoring the chromatographic behavior of the target peptide photo releasable biotin affinity tag for selective capture and release of tagged peptide and heavy isotope tag for differentiating analyte from internal standards these reagents are used together with isoelectric focusing and reverse phase microbore chromatography electrospray ionization tandem mass spectrometry to determine the absolute abundance of set of target protein in complex mixture such as cell lysate VICAT reagents should also be useful for detecting low abundance protein in biological fluids such as serum and for the detection of posttranslational protein modifications and different splice variants

one of the primary goals of proteomics is the description of the composition dynamics and connections of the multiprotein modules that catalyze wide range of biological functions in cell mass spectrometry MS has proven to be an extremely powerful tool for characterizing the composition of purified complexes however because MS is not quantitative technique the usefulness of the data is limited for example without quantitative measurements it is difficult to detect dynamic changes in complex composition and it can be difficult to distinguish bona fide complex components from nonspecifically copurifying protein In this chapter we describe strategy for characterizing the composition of protein complexes and their dynamic changes in composition by combining affinity purification approaches with stable isotope tagging and MS the use of software tools for statistical analysis of the data is also described

We have developed novel androgen receptor AR expression system in the human embryonic kidney cell line that recapitulates AR biochemical activity as steroid hormone receptor in prostate cancer cell We used this system to identify putative AR binding protein in the cytosolic and nuclear compartments of mammalian cell using large scale co immunoprecipitation strategy coupled to quantitative mass spectrometry for example the heat shock and chaperones which are known regulators of steroid hormone receptor were identified as AR binding protein AR purification enriched for protein involved in RNA processing protein transport and cytoskeletal organization suggesting functional link between AR and these protein modules in mammalian cell for example AR purification in the nuclear compartment led to the specific enrichment of actinin clathrin heavy chain and serine threonine protein kinase short interfering RNA knockdown studies and co transcriptional reporter assay revealed that clathrin heavy chain possessed co activator activity during AR mediated transcription whereas actinin and protein kinase displayed both co activator and co repressor activity during AR mediated transcription that was dependent upon their relative expression levels lastly immunohistochemical staining of prostate tissue showed that actinin levels decreased in the nucleus of high grade cancerous prostate samples suggesting its possible deregulation in advanced prostate cancers as previously observed in late stage metastatic breast cancers taken together these findings suggest AR binds to specific protein modules in mammalian cell and that these protein modules may provide molecular framework for interrogating AR function in normal and cancerous prostate epithelial cell

mass spectrometry based quantitative proteomics has become an important component of biological and clinical research although such analyses typically assume that protein peptide fragments are observed with equal likelihood only few so called proteotypic peptide are repeatedly and consistently identified for any given protein present in mixture using peptide identifications generated by four proteomic platforms we empirically identified proteotypic peptide for distinct yeast protein characteristic physicochemical properties of these peptide were used to develop computational tool that can predict proteotypic peptide for any protein from any organism for given platform with cumulative accuracy possible applications of proteotypic peptide include validation of protein identifications absolute quantification of protein annotation of coding sequence in genomes and characterization of the physical principles governing key elements of mass spectrometric workflows digestion chromatography ionization and fragmentation

It has long been thought that blood plasma could serve as window into the state of one organs in health and disease because tissue derived protein represent significant fraction of the plasma proteome although substantial technical progress has been made toward the goal of comprehensively analyzing the blood plasma proteome the basic assumption that protein derived from variety of tissues could indeed be detectable in plasma using current proteomics technologies has not been rigorously tested here we provide evidence that such tissue derived protein are both present and detectable in plasma via direct mass spectrometric analysis of captured glycopeptides and thus provide conceptual basis for plasma protein biomarker discovery and analysis

OBJECTIVES pancreatitis is an inflammatory condition of the pancreas however it often shares many molecular features with pancreatic cancer biomarkers present in pancreatic cancer frequently occur in the setting of pancreatitis the efforts to develop diagnostic biomarkers for pancreatic cancer have thus been complicated by the false positive involvement of pancreatitis method In an attempt to develop protein biomarkers for pancreatic cancer we previously use quantitative proteomics to identify and quantify the protein from pancreatic cancer juice pancreatic juice is rich source of protein that are shed by the pancreatic ductal cell In this study we used similar approach to identify and quantify protein from pancreatitis juice RESULTS In total protein were identified and quantified in the comparison of pancreatic juice from pancreatitis patients versus pooled normal control juice nineteen of the juice protein were overexpressed and were underexpressed in pancreatitis juice by at least fold compared with normal pancreatic juice Of these differentially expressed protein in pancreatitis protein were also differentially expressed in the pancreatic juice from pancreatic cancer patient CONCLUSIONS identification of these differentially expressed protein from pancreatitis juice provides useful information for future study of specific pancreatitis associated protein and to eliminate potential false positive biomarkers for pancreatic cancer

quantitative profiling of protein the direct effectors of nearly all biological functions will undoubtedly complement technologies for the measurement of mRNA systematic proteomic measurement of the cell cycle is now possible by using stable isotopic labeling with isotope coded affinity tag reagents and software tools for high throughput analysis of LC MS MS data We provide here the first such study achieving quantitative global proteomic measurement of time course gene expression experiment in model eukaryote the budding yeast saccharomyces cerevisiae during the cell cycle We sampled of all predicted ORFs and provide the data including identifications quantitations and statistical measures of certainty to the community in sortable matrix We do not detect significant concordance in the dynamics of the system over the time course tested between our proteomic measurements and microarray measures collected from similarly treated yeast cultures our proteomic dataset therefore provides necessary and complementary measure of eukaryotic gene expression establishes rich database for the functional analysis of cerevisiae protein and will enable further development of technologies for global proteomic analysis of higher eukaryotes

this chapter describes method for site specific stable isotope labeling of cysteinyl peptide in complex peptide mixtures via solid phase capture and release process and the concomitant isolation of the labeled peptide relative quantification of protein from different samples by mass spectrometry MS is based on the stable isotope dilution approach the isotopically labeled peptide are combined purified or separated into fractions and analyzed by mass spectrometry which measures the mass and ion abundance of peptide because isotopically heavy and light forms of peptide of the same amino-acid sequence are chemically identical they generate responses with identical sensitivity from the mass spectrometer and are readily distinguished based on their mass differences for labeling with SH specific solid phase reagents it is advantageous to label peptide instead of protein because protein may possess tertiary structures that render some cysteine residue inaccessible to the solidphase reagent protein digestion can be performed with any commercially available and suitable protease

the proteomes most likely to contain clinically useful disease biomarkers are those of human body fluids three recent large scale proteomic analyses of tears urine and seminal plasma using the latest mass spectrometric technology will provide useful datasets for biomarker discovery

We present the saccharomyces cerevisiae peptideatlas composed from diverse experiments and million tandem mass spectra the observed peptide align to of saccharomyces genome database SGD open reading frames ORFs of the uncharacterized SGD ORFs of cerevisiae ORFs with gene ontology annotation of molecular function unknown and of ORFs with gene names We highlight the use of this resource for data mining construction of high quality lists for targeted proteomics validation of protein and software development

accurate consistent and transparent data processing and analysis are integral and critical parts of proteomics workflows in general and for biomarker discovery in particular definition of common standards for data representation and analysis and the creation of data repositories are essential to compare exchange and share data within the community current issues in data processing analysis and validation are discussed together with opportunities for improving the process in the future and for defining alternative workflows

this article describes the origins working practices and various development projects of the Human proteome organisation proteomics standards initiative HUPO PSI specifically our work on reporting requirements data exchange formats and controlled vocabulary terms We also offer our view of the two functional genomics projects in which the PSI plays role FuGE and FuGO discussing their impact on our process and laying out the benefits we see as accruing both to the PSI and to biomedical science as whole as result of their widespread acceptance

multidimensional LC MS based shotgun proteomics experiments at the peptide level have traditionally been carried out by ion exchange in the first dimension and reversed phase liquid chromatography in the second recently it has been shown that isoelectric focusing IEF is an interesting alternative approach to ion exchange separation of peptide in the first dimension here we present an improved protocol for peptide separation by continuous free flow electrophoresis FFE as the first dimension in two dimensional peptide separation work flow By the use of flat gradient and mannitol and urea based separation media we were able to perform high throughput proteome analysis with improved interfacing between FFE and RPLC MS MS the developed protocol was applied to cytosolic fraction from schneider S2 cell from drosophila melanogaster resulting in the identification of more than unique peptide with high probability To improve the accuracy of the peptide identification following FFE IEF we incorporated the information as an additional parameter into statistical model for discrimination between correct and incorrect peptide assignments to MS MS spectra

distance constraints in protein and protein complexes provide invaluable information for calculation of 3D structures identification of protein binding partners and localization of protein protein contact sites We have developed an integrative approach to identify and characterize such sites through the analysis of proteolytic products derived from protein chemically cross linked by isotopically coded cross linkers using LC MALDI tandem mass spectrometry and computer software this method is specifically tailored toward the rapid analysis of low microgram amounts of protein or multimeric protein complexes cross linked with nonlabeled and deuterium labeled bis NHS ester cross linking reagents both commercially available and readily synthesized through labeling with 18O water solvent and LC MALDI analysis the method further allows the possible distinction between type and type or type modified peptide monolinks and looplinks or cross links although such distinction is more readily made from analysis of tandem mass spectrometry data when applied to the bacterial colicin E7 DNase lm7 heterodimeric protein complex cross links were identified including six intersubunit cross links all between residue that are close in space when examined in the context of the ray structure of the heterodimer In addition cross links were successfully identified in five single subunit protein beta lactoglobulin cytochrome lysozyme myoglobin and ribonuclease establishing the generality of the approach

using chemical genetics screen we have identified ent oxokaurenoic acid EKA as chemical that causes prolonged mitotic arrest at stage resembling prometaphase EKA inhibits the association of the mitotic motor protein centromeric protein with kinetochores and inhibits chromosome movement unlike most antimitotic agents EKA does not inhibit the polymerization or depolymerization of tubulin To identify EKA interacting protein we used cell permeable biotinylated form that retains biological activity to isolate binding protein from living cell mass spectrometric analysis identified six EKA binding protein including ran binding protein kinetochore protein whose depletion by small interfering RNA causes similar mitotic arrest phenotype

there has been considerable recent interest in proteomic analyses of plasma for the purpose of discovering biomarkers profiling linked glycopeptides is particularly promising method because the population of linked glycosites represents the proteomes of plasma the cell surface and secreted protein at very low redundancy and provides compelling link between the tissue and plasma proteomes here we describe unipep http www unipep org database of human linked glycosites as resource for biomarker discovery

protein glycosylation has long been recognized as very common posttranslational modification protein glycosylation is prevalent in protein destined for extracellular environments these include protein localized on the extracellular surface and those secreted to body fluids In search of method that has the potential to identify and quantify most protein found in body fluids or the cell surface we have recently developed novel method for solid phase extraction of formerly linked glycosylated peptide from glycoproteins It has been shown that protein secreted to body fluids or localized on the cell surface can be specifically enriched by this method the technique is based on the conjugation of glycoproteins to solid support using hydrazide chemistry removal of nonglycosylated peptide by trypsin digestion stable isotope labeling of glycopeptides and the specific release of formerly linked glycosylated peptide via peptide glycosidase the recovered formerly linked glycopeptides are then identified and quantified by tandem mass spectrometry

pancreatic juice is an exceptionally rich source of cancer specific protein shed from cancerous ductal cell into the pancreatic juice quantitative proteomic analysis of the protein specific to pancreatic cancer juice has not previously been reported We used isotope code affinity tag ICAT technology and MS MS to perform quantitative protein profiling of pancreatic juice from pancreatic cancer patients and normal controls ICAT technology coupled with MS MS allows the systematic study of the proteome and measures the protein abundance in pancreatic juice with the potential for development of biomarkers total of protein were identified and quantified in the pancreatic juice from pancreatic cancer patient of which protein showed abundance changes of at least twofold in pancreatic cancer juice compared to normal controls many of these protein have been externally validated this is the first comprehensive study of the pancreatic juice proteome by quantitative global protein profiling and the study reveals numerous protein that are shown for the first time to be associated with pancreatic cancer providing candidates for diagnostic biomarkers one of the identified protein insulin like growth factor binding protein was further validated by western blotting to be elevated in pancreatic cancer juice and overexpressed in pancreatic cancer tissue

tandem mass spectrometry MS MS allows for the rapid identification of many types of post translational modifications PTMs especially those that can be detected by diagnostic mass shift in one or more peptide fragment ions for example phosphorylation but some PTMs for example SUMOs and other ubiquitin like modifiers themselves produce multiple fragment ions combined with fragments from the modified target peptide complex overlapping fragmentation pattern is thus generated which is uninterpretable by standard peptide sequencing software here we introduce SUMmOn an automated pattern recognition tool that detects diagnostic PTM fragment ion series within complex MS MS spectra to identify modified peptide and modification sites within these peptide using SUMmOn we demonstrate for the first time that human SUMO multimerizes in vitro primarily via three terminal lysines lys7 lys16 and lys17 notably our method is theoretically applicable to any type of modification or chemical moiety generating unique fragment ion pattern

ligation of the cell antigen receptor BCR with antigen induces lipid raft coalescence process that occurs after crosslinking of variety of signaling receptors and is thought to potentiate cellular activation To investigate lipid raft dynamics during BCR signaling we quantitatively analyzed the cell lipid raft proteome BCR engagement induced dissociation of the adaptor protein ezrin from lipid rafts as well as threonine dephosphorylation of ezrin and its concomitant detachment from actin indicating transient uncoupling of lipid rafts from the actin cytoskeleton expression of constitutively active ezrin chimeras inhibited the BCR induced coalescence of lipid rafts our data demonstrate that the release of ezrin from lipid rafts acts as critical trigger that regulates lipid raft dynamics during BCR signaling

A main objective of proteomics research is to systematically identify and quantify protein in given proteome cell subcellular fractions protein complexes tissues or body fluids protein labeling with isotope coded affinity tags ICAT followed by tandem mass spectrometry allows sequence identification and accurate quantification of protein in complex mixtures and has been applied to the analysis of global protein expression changes protein changes in subcellular fractions components of protein complexes protein secretion and body fluids this protocol describes protein sample labeling with ICAT reagents chromatographic fractionation of the ICAT labeled tryptic peptide and protein identification and quantification using tandem mass spectrometry the method is suitable for both large scale analysis of complex samples including whole proteomes and small scale analysis of subproteomes and allows quantitative analysis of protein including those that are difficult to analyze by gel based proteomics technology

To better understand the extremely halophilic archaeon halobacterium species NRC we analyzed its soluble proteome by two dimensional liquid chromatography coupled to electrospray ionization tandem mass spectrometry total of unique protein were identified with protein prophet probability between and To evaluate the biochemical activities of the organism the proteomic data were subjected to biological network analysis using our BMsorter software this allowed us to examine the protein expressed in different biomodules and study the interactions between pertinent biomodules interestingly an integrated analysis of the enzymes in the amino-acid metabolism and citrate cycle networks suggested that up to eight amino-acid may be converted to oxaloacetate fumarate or oxoglutarate in the citrate cycle for energy production In addition glutamate and aspartate may be interconverted from other amino-acid or synthesized from citrate cycle intermediates to meet the high demand for the acidic amino-acid that are required to build the highly acidic proteome of the organism thus this study demonstrated that proteome analysis can provide useful information and help system analyses of organisms by the american society for biochemistry and molecular biology inc

We identified the gene encoding chondroitin glucuronate C5 epimerase EC that converts glucuronic acid to iduronic acid residue in dermatan sulfate biosynthesis the enzyme was solubilized from bovine spleen and an fold purified preparation containing major kDa candidate component was subjected to mass spectrometry analysis of tryptic peptide SART2 squamous cell carcinoma antigen recognized by cell protein with unknown function highly expressed in cancer cell and tissues was identified by peptide covering of the sequence transient expression of cDNA resulted in fold increase in epimerase activity in 293HEK cell lysate moreover overexpressing cell produced dermatan sulfate chains with of iduronic acid containing disaccharide units as compared with for mock transfected cell the iduronic acid residue were preferentially clustered in blocks as in naturally occurring dermatan sulfate given the discovered identity we propose to rename SART2 nakao shichijo imaizumi inoue matsunaga yamada kikuchi tsuda ohta takamori yamana fujita and itoh immunol with functional designation chondroitin glucuronate C5 epimerase or DS epimerase DS epimerase activity is ubiquitously present in normal tissues although with marked quantitative differences It is highly homologous to part of the NCAG1 protein encoded by the C18orf4 gene genetically linked to bipolar disorder NCAG1 also contains putative chondroitin sulfate sulfotransferase domain and thus may be involved in dermatan sulfate biosynthesis the functional relation between dermatan sulfate and cancer is unknown but may involve known iduronic acid dependent interactions with growth factors selectins cytokines or coagulation inhibitors

mass spectrometry is central analytical technique for protein research and for the study of biomolecules in general driven by the need to identify characterize and quantify protein at ever increasing sensitivity and in ever more complex samples wide range of new mass spectrometry based analytical platforms and experimental strategies have emerged here we review recent advances in mass spectrometry instrumentation in the context of current and emerging research strategies in protein science

In mass spectrometry based proteomics frequently hundreds of thousands of MS MS spectra are collected in single experiment Of these relatively small fraction is confidently assigned to peptide sequence whereas the majority of the spectra are not further analyzed spectra are not assigned to peptide for diverse reasons these include deficiencies of the scoring schemes implemented in the database search tools sequence variations single nucleotide polymorphisms or omissions in the database searched post translational or chemical modifications of the peptide analyzed or the observation of sequence that are not anticipated from the genomic sequence splice forms somatic rearrangement and processed protein To increase the amount of information that can be extracted from proteomic MS MS datasets we developed robust method that detects high quality spectra within the fraction of spectra unassigned by conventional sequence database searching and computes quality score for each spectrum We also demonstrate that iterative search strategies applied to such detected unassigned high quality spectra significantly increase the number of spectra that can be assigned from datasets and that biologically interesting new insights can be gained from existing data

enteropathogenic escherichia coli EPEC is an enteric human pathogen responsible for much worldwide morbidity and mortality EPEC uses type III secretion system to inject bacterial protein into the cytosol of intestinal epithelial cell to cause diarrheal disease We are interested in determining the host protein to which EPEC translocator and effector protein bind during infection To facilitate protein enrichment we created fusions between GST and EPEC virulence protein and expressed these fusions individually in saccharomyces cerevisiae the biology of cerevisiae is well understood and often employed as model eukaryote to study the function of bacterial virulence factors We isolated the yeast protein that interact with individual EPEC protein by affinity purifying against the GST tag these complexes were subjected to ICAT combined with ESI MS MS database searching of sequenced peptide provided list of protein that bound specifically to each EPEC virulence protein the dataset suggests several potential mammalian targets of these protein that may guide future experimentation

mass spectrometry based proteomic experiments in combination with liquid chromatography based separation can be used to compare complex biological samples across multiple conditions these comparisons are usually performed on the level of protein lists generated from individual experiments unfortunately given the current technologies these lists typically cover only small fraction of the total protein content making global comparisons extremely limited recently approaches have been suggested that are built on the comparison of computationally built feature lists instead of protein identifications although these approaches promise to capture bigger spectrum of the protein present in complex mixture their success is strongly dependent on the correctness of the identified features and the aligned retention times of these features across multiple experiments In this experimental computational study we went one step further and performed the comparisons directly on the signal level first signal maps were constructed that associate the experimental signals across multiple experiments then feature detection algorithm used this integrated information to identify those features that are discriminating or common across multiple experiments At the core of our approach is score function that faithfully recognizes mass spectra from similar peptide mixtures and an algorithm that produces an optimal alignment time warping of the liquid chromatography experiments on the basis of raw MS signal making minimal assumptions on the underlying data We provide experimental evidence that suggests uniqueness and correctness of the resulting signal maps even on low accuracy mass spectrometers these maps can be used for variety of proteomic analyses here we illustrate the use of signal maps for the discovery of diagnostic biomarkers An implementation of our algorithm is available on our web server

myc is key regulatory protein in higher eukaryotes controlling important cellular functions such as proliferation differentiation and apoptosis myc is profoundly involved in the genesis of many human and animal cancers and the abrogation of myc induced apoptosis is critical event in cancer progression because the mechanisms that mediate myc induced apoptosis are largely unknown we analyzed protein expression during myc induced apoptosis using an isotope code daffinity tag quantitative proteomics approach and identified that proapoptotic mitochondrial chloride ion channel mtCLIC CLIC4 is induced by myc myc binds to the mtCLIC gene promoter and activates its transcription suppression of mtCLIC expression by RNA interference inhibited myc induced apoptosis in response to different stress conditions and abolished the cooperative induction of apoptosis by myc and bax We also found that myc reduces the expression of bcl and bcl xL and that the apoptosis inducing stimuli up regulate bax expression these results suggest that up regulation of mtCLIC together with reduction in bcl and bcl xL sensitizes myc expressing cell to the proapoptotic action of bax

the autumn workshop of the proteomics standards initiative of the human proteomics organisation met to further advance the development of the existing standards in the fields of molecular interactions and mass spectrometry In addition new areas were addressed in particular developing standards for the description and exchange of data from gel electrophoresis experiments the general proteomics standards group is now working closely with the FuGE functional genomics experiment efforts to define general standard in which to encode data that will enable system biology approach to data analysis common to all these efforts is the field of protein modifications and work has been initiated to establish an ontology in this field that can be used by both workers in the field of proteomics and the wider scientific community

the transcriptional corepressor msin3 is associated with histone deacetylases HDACs and is utilized by many DNA binding transcriptional repressors We have cloned and characterized novel msin3A binding protein SAP25 SAP25 binds to the PAH1 domain of msin3A associates with the msin3A HDAC complex in vivo and represses transcription when tethered to DNA SAP25 is required for msin3A mediated but not CoR mediated repression SAP25 is nucleocytoplasmic shuttling protein actively exported from the nucleus by CRM1 dependent mechanism fraction of SAP25 is located in promyelocytic leukemia protein PML nuclear bodies and PML induces striking nuclear accumulation of SAP25 An isotope coded affinity tag quantitative proteomic analysis of the SAP25 complex revealed that SAP25 is associated with several components of the msin3 complex nuclear export machinery and regulators of transcription and cell cycle these results suggest that SAP25 is novel core component of the msin3 corepressor complex whose subcellular location is regulated by PML copyright

one of the most important roles that mass spectrometry MS has played in the late twentieth and early twenty first centuries has been to assist in the growth of knowledge of dynamic phosphorylation events not only has MS allowed researches to pinpoint the site of phosphorylation but it has also enabled them to identify the kinase phosphatase pairs responsible for regulation of specific modification as well as to follow the functional consequences of the observed phosphorylation events on the biology of the system for phosphorylation analysis the important contribution of MS has been critical but not definitive there are numerous method that have been applied with success yet none are generally applicable to all analyses So for the time being researchers in the field must select from panel of method to find de phosphorylation events In the work described in this chapter collection of integrated method are presented detailed account is provided for phosphorylation capture via on and off line immobilized metal affinity chromatography IMAC this is followed by suite of useful strategies for discovery of phosphorylation positioning through sequence determination by phosphate specific diagnostic ion scans including precursor and product ion scans neutral loss scans and in source dissociation and post source decay copyright elsevier inc all rights reserved

the completion of the sequencing of the human genome and the concurrent rapid development of high throughput proteomic method have resulted in an increasing need for automated approaches to archive proteomic data in repository that enables the exchange of data among researchers and also accurate integration with genomic data peptideatlas http www peptideatlas org addresses these needs by identifying peptide by tandem mass spectrometry MS MS statistically validating those identifications and then mapping identified sequence to the genomes of eukaryotic organisms meaningful comparison of data across different experiments generated by different groups using different types of instruments is enabled by the implementation of uniform analytic process this uniform statistical validation ensures consistent and high quality set of peptide and protein identifications the raw data from many diverse proteomic experiments are made available in the associated peptideatlas repository in several formats here we present summary of our process and details about the human drosophila and yeast peptideatlas builds

In the field of proteomics reproducible liquid chromatographic description of analytes is often key element for the differentiation or identification of protein or peptide for clinical or biological research projects however analyte identification by retention time can be problematic in proteomics where lack of standardization can result in significantly different chromatography for the same analytes analyzed on different machines here we present novel method of monitoring the mobile phase gradient of LC MS MS analyses by monitoring the ion current signal intensities of tracer molecules dissolved in the mobile phase solvents the tracers ion current signal intensities chronicled gradient fluctuations did not adversely affect the number or quality of CID based sequence identifications and had lower run to run variance when compared to retention time

the analysis of tandem mass MS MS data to identify and quantify protein is hampered by the heterogeneity of file formats at the raw spectral data peptide identification and protein identification levels different mass spectrometers output their raw spectral data in variety of proprietary formats and alternative method that assign peptide to MS MS spectra and infer protein identifications from those peptide assignments each write their results in different formats here we describe an MS MS analysis platform the trans proteomic pipeline which makes use of open XML file formats for storage of data at the raw spectral data peptide and protein levels this platform enables uniform analysis and exchange of MS MS data generated from variety of different instruments and assigned peptide using variety of different database search programs We demonstrate this by applying the pipeline to data sets generated by thermofinnigan LCQ ABI MALDI TOF TOF and waters TOF instruments and searched in turn using SEQUEST mascot and COMET

the plenary session of the proteomics standards initiative of the human proteome organisation discussed the current status of the ongoing work in the fields of molecular interactions mass spectrometry and the description of protein modifications In addition new areas are being opened up in particular developing standards for the description and exchange of data from gel electrophoresis experiments the general proteomics standards group is now working closely with the functional genomics experiment efforts to define general standard in which to encode data that will enable system biology approach to data analysis

In MS MS experiments with automated precursor ion selection only fraction of sequencing attempts lead to the successful identification of peptide number of reasons may contribute to this situation they include poor fragmentation of the selected precursor ion the presence of modified residue in the peptide mismatches with sequence databases and frequently the concurrent fragmentation of multiple precursors in the same CID attempt current database search engines are incapable of correctly assigning the sequence of multiple precursors to such spectra We have developed search engine probIdtree which can identify multiple peptide from CID spectrum generated by the concurrent fragmentation of multiple precursor ions this is achieved by iterative database searching in which the submitted spectra are generated by subtracting the fragment ions assigned to tentatively matched peptide from the acquired spectrum and in which each match is assigned tentative probability score tentatively matched peptide are organized in tree structure from which their adjusted probability scores are calculated and used to determine the correct identifications the results using MALDI TOF TOF MS MS data demonstrate that multiple peptide can be effectively identified simultaneously with high confidence using probIdtree

A very popular approach in proteomics is the so called shotgun LC MS MS strategy In its mostly used form total protein digest is separated by ion exchange fractionation in the first dimension followed by off or on line RP LC MS MS We replaced the first dimension by isoelectric focusing in the liquid phase using the off gel device producing fractions As peptide are separated by their isoelectric point in the first dimension and hydrophobicity in the second those experimentally derived parameters pI and can be used for the validation of potentially identified peptide We applied this strategy to cellular extract of drosophila Kc167 cell and identified peptide with two different database search engines namely PHENYX and SEQUEST with peptideprophet validation of the SEQUEST results PHENYX returned potential peptide identifications and SEQUEST the SEQUEST results were reduced to identifications by validation with peptideprophet validation of the peptideprophet SEQUEST and PHENYX results by pI and parameters confirmed peptideprophet identifications while in the remainder of the SEQUEST results another peptide were found to be likely hits the validation on PHENYX resulted in the fixation of solid value threshold of lt that sets by itself the correct identification confidence to gt and final count of highly confident peptide identifications was achieved after pI and validation although the peptideprophet and PHENYX datasets have very high confidence the overlap of common identifications was only at to be explained by the fact that data interpretation was done searching different protein databases with two search engines of different algorithms the approach used in this study allowed for an automated and improved data validation process for shotgun proteomics projects producing MS MS peptide identification results of very high confidence

using combination of tandem affinity purification tagging and mass spectrometry we characterized novel evolutionarily conserved protein phosphatase PP4 containing complex PP4cs protein phosphatase cisplatin sensitive complex that plays critical role in the eukaryotic DNA damage response PP4cs is comprised of the catalytic subunit PP4C known regulatory subunit PP4R2 and novel protein that we termed PP4R3 the saccharomyces cerevisiae PP4R3 ortholog psy2 was identified previously in screen for sensitivity to the DNA damaging agent and anticancer drug cisplatin We demonstrated that deletion of any of the PP4cs complex orthologs in cerevisiae elicited cisplatin hypersensitivity furthermore human PP4R3 complemented the yeast psy2 deletion and drosophila melanogaster lacking functional PP4R3 flfl exhibited cisplatin hypersensitivity suggesting highly conserved role for PP4cs in DNA damage repair finally we found that PP4R3 may target PP4cs to the DNA damage repair machinery at least in part via an interaction with rad53 CHK2

We present method for peptide and protein identification based on LC MS profiling the method identified peptide at high throughput without expending the sequencing time necessary for CID spectra based identification the measurable peptide properties of mass and liquid chromatographie elution conditions are used to characterize and differentiate peptide features and these peptide features are matched to reference database from previously acquired and archived LC MS MS experiments to generate sequence assignments the matches are scored according to the probability of an overlap between the peptide feature and the database peptide resulting in ranked list of possible peptide sequence for each peptide submitted this method resulted in times more peptide sequence identifications from single LC MS analysis of yeast than from shotgun peptide sequencing using LC MS MS

A crucial aim upon the completion of the human genome is the verification and functional annotation of all predicted gene and their protein products here we describe the mapping of peptide derived from accurate interpretations of protein tandem mass spectrometry MS data to eukaryotic genomes and the generation of an expandable resource for integration of data from many diverse proteomics experiments furthermore we demonstrate that peptide identifications obtained from high throughput proteomics can be integrated on large scale with the human genome this resource could serve as an expandable repository for MS derived proteome information

skeletal muscle atrophy is process in which protein degradation exceeds protein synthesis resulting in decrease of the muscle physiological cross sectional area and mass and is often serious consequence of numerous health problems We used the isotope coded affinity tag ICAT labelling approach and MS MS to protein profile cytosolic subcellular fractions from mouse tibialis anterior skeletal muscle undergoing or days of immobilisation induced atrophy for the validation of peptide and protein identifications statistical algorithms were applied to the sequence database search results in order to obtain consistent sensitivity error rates for protein and peptide identifications at each immobilisation time point In this study we identified and quantified large number of mouse skeletal muscle protein At protein probability of corresponding to false positive error rate of less than protein were identified for days of immobilisation and for the control sample respectively from which displayed altered protein abundance with atrophy due to randomness of data acquisition full time course could be generated only for protein most of which displayed unchanged protein abundance In spite of this useful information about dataset characteristics and underlying biological process could be obtained through gene over representation analysis gene categories mainly but not exclusively encoded by the subset of overlapping protein were consistently found to be significantly over represented in all sub datasets

the shotgun proteomic strategy based on digesting protein into peptide and sequencing them using tandem mass spectrometry and automated database searching has become the method of choice for identifying protein in most large scale studies however the peptide centric nature of shotgun proteomics complicates the analysis and biological interpretation of the date especially in the case of higher eukaryote organisms the same peptide sequence can be present in multiple different protein or protein isoforms such shared peptide therefore can lead to ambiguities in determining the identities of sample protein In this article we illustrate the difficulties of interpreting shotgun proteomic data and discuss the need for common nomenclature and transparent informatic approaches We also discuss related issues such as the state of protein sequence databases and their role in shotgun proteomic analysis interpretation of relative peptide quantification data in the presence of multiple protein isoforms the integration of proteomic and transcriptional data and the development of computational infrastructure for the integration of multiple diverse datasets

background aims pancreatic cancer is highly lethal disease that has seen little headway in diagnosis and treatment for the past few decades the effective treatment of pancreatic cancer is critically relying on the diagnosis of the disease at an early stage which still remains challenging new experimental approaches such as quantitative proteomics have shown great potential for the study of cancer and have opened new opportunities to investigate crucial events underlying pancreatic tumorigenesis and to exploit this knowledge for early detection and better intervention method To systematically study protein expression in pancreatic cancer we used isotope coded affinity tag technology and tandem mass spectrometry to perform quantitative proteomic profiling of pancreatic cancer tissues and normal pancreas results total of protein were identified and quantified in pancreatic cancer samples of which were differentially expressed in cancer by at least fold this study revealed numerous protein that are newly discovered to be associated with pancreatic cancer providing candidates for future early diagnosis biomarkers and targets for therapy several differentially expressed protein were further validated by tissue microarray immunohistochemistry many of the differentially expressed protein identified are involved in protein driven interactions between the ductal epithelium and the extracellular matrix that orchestrate tumor growth migration angiogenesis invasion metastasis and immunologic escape conclusions our study is the first application of isotope coded affinity tag technology for proteomic analysis of human cancer tissue and has shown the value of this technology in identifying differentially expressed protein in cancer

spa2p is nonessential protein that regulates yeast cell polarity It localizes early to the presumptive bud site and remains at sites of growth throughout the cell cycle To understand how spa2p localization is regulated and to gain insight into its molecular function in cell polarity we used coimmunoprecipitation strategy followed by tandem mass spectrometry analysis to identify protein that associate with spa2p in vivo We identified myo1p myo2p pan1p and the protein encoded by YFR016c as protein that interact with spa2p strikingly all of these protein are involved in cell polarity and or actin function here we focus on the functional significance of the interactions of spa2p with myo2p and myo1p We find that localization of spa2GFP to sites of polarized growth depends on functional myo2p but not on myo1p We also find that spa2p like myo2p cosediments with actin in an ATP sensitive manner We hypothesize that spa2p associates with actin via direct or indirect interaction with myo2p and that spa2p may be involved in mediating polarized localization of polarity protein via myo2p In addition we observe an enhanced cell separation defect in myo1spa2 strain at this provides further evidence that spa2p is involved in cytokinesis and cell wall morphogenesis

there is an increasing interest in the quantitative proteomic measurement of the protein contents of substantially similar biological samples for the analysis of cellular response to perturbations over time or for the discovery of protein biomarkers from clinical samples technical limitations of current proteomic platforms such as limited reproducibility and low throughput make this challenging task new LC MS based platform is able to generate complex peptide patterns from the analysis of proteolyzed protein samples at high throughput and represents promising approach for quantitative proteomics crucial component of the LC MS approach is the accurate evaluation of the abundance of detected peptide over many samples and the identification of peptide features that can stratify samples with respect to their genetic physiological or environmental origins We present here new software suite specarray that generates peptide versus sample array from set of LC MS data peptide array stores the relative abundance of thousands of peptide features in many samples and is in format identical to that of gene expression microarray peptide array can be subjected to an unsupervised clustering analysis to stratify samples or to discriminant analysis to identify discriminatory peptide features We applied the specarray to analyze two sets of LC MS data one was from four repeat LC MS analyses of the same glycopeptide sample and another was from LC MS analysis of serum samples of five male and five female mice We demonstrate through these two study cases that the specarray software suite can serve as an effective software platform in the LC MS approach for quantitative proteomics

We present robust and general method for the identification and relative quantification of phosphorylation sites in complex protein mixtures It is based on new chemical derivatization strategy using dendrimer as soluble polymer support and tandem mass spectrometry MS MS In single step phosphorylated peptide are covalently conjugated to dendrimer in reaction catalyzed by carbodiimide and imidazole modified phosphopeptides are released from the dendrimer via acid hydrolysis and analyzed by MS MS when coupled with an initial antiphosphotyrosine protein immunoprecipitation step and stable isotope labeling in single experiment we identified all known tyrosine phosphorylation sites within the immunoreceptor tyrosine based activation motifs ITAM of the cell receptor TCR CD3 chains and previously unknown phosphorylation sites on total tyrosine phosphoproteins and their interacting partners in human cell the dynamic changes in phosphorylation were quantified in these protein

peptide identifications of high probability from LC MS MS human serum and plasma experiments from eight different laboratories carried out in the context of the HUPO plasma proteome project were combined and mapped to the ensEMBL human genome the distinct observed peptide were mapped to approximately different protein the resulting compendium of peptide and their associated samples protein and gene is made publicly available as reference for future research on human plasma

HUPO initiated the plasma proteome project PPP in its pilot phase has evaluated advantages and limitations of many depletion fractionation and MS technology platforms compared PPP reference specimens of human serum and EDTA heparin and citrate anticoagulated plasma and created publicly available knowledge base www bioinformatics med umich edu hupo ppp www ebi ac uk pride thirty five participating laboratories in countries submitted datasets working groups addressed specimen stability and protein concentrations protein identifications from MS MS datasets independent analyses from raw MS MS spectra search engine performance subproteome analyses and biological insights antibody arrays and direct MS SELDI analyses MS MS datasets had different international protein index IPI protein IDs our integration algorithm applied to multiple matches of peptide sequence yielded IPI protein identified with one or more peptide and protein identified with two or more peptide the core dataset these protein have been characterized with gene ontology interpro novartis atlas OMIM and immunoassay based concentration determinations the database permits examination of many other subsets such as protein identified with three or more peptide reverse protein to DNA matching identified protein for previously unidentified ORFs We recommend use of plasma instead of serum with EDTA or citrate for anticoagulation To improve resolution sensitivity and reproducibility of peptide identifications and protein matches we recommend combinations of depletion fractionation and MS MS technologies with explicit criteria for evaluation of spectra use of search algorithms and integration of homologous protein matches this special issue of PROTEOMICS presents papers integral to the collaborative analysis plus many reports of supplementary work on various aspects of the PPP workplan these PPP results on complexity dynamic range incomplete sampling false positive matches and integration of diverse datasets for plasma and serum protein lay foundation for development and validation of circulating protein biomarkers in health and disease

MS MS fragmentation of peptide is dominated by overlapping and ion series however alternative fragmentation possibilities exist including neutral loss database was generated containing MS MS spectra of tryptic peptide assigned with high probability to an amino-acid sequence true positives and set of certified false true negative assignments for analysis of the amino terminus similar database was created for analysis of neutral loss at the carboxy termini using data set of chymotryptic peptide the analysis demonstrated that the presence of an internal basic residue limiting proton mobility has profound effect on neutral loss peptide with fully mobile protons demonstrated minimal neutral loss with the exception of amide bonds with proline on the carboxy terminal side which created an intense neutral loss peak In contrast peptide with partial proton mobility contained many amino-acid on either side of the amide bond associated with strong neutral loss peak most notable among these was proline on the carboxy terminal side of an amide bond and aspartic acid on the amino terminal side of bond all results were found to be consistent for doubly and triply charged peptide and after adjustment for pairings across the amide bonds with particularly labile residue the carboxy terminal of chymotryptic peptide also demonstrated significant neutral loss events associated with numerous amino-acid residue clarification of the rules that govern neutral loss when incorporated into analysis software will improve our ability to correctly assign spectra to peptide sequence

synaptosomes are isolated synapses produced by subcellular fractionation of brain tissue they contain the complete presynaptic terminal including mitochondria and synaptic vesicles and portions of the postsynaptic side including the postsynaptic membrane and the postsynaptic density PSyD proteomic characterisation of synaptosomes isolated from mouse brain was performed employing the isotope coded affinity tag ICAT method and tandem mass spectrometry MS MS after isotopic labelling and tryptic digestion peptide were fractionated by cation exchange chromatography and cysteine containing peptide were isolated by affinity chromatography the peptide were identified by microcapillary liquid chromatography electrospray ionisation MS MS µLC ESI MS MS In two experiments peptide representing total of database entries were identified they are involved in different presynaptic and postsynaptic functions including synaptic vesicle exocytosis for neurotransmitter release vesicle endocytosis for synaptic vesicle recycling as well as postsynaptic receptors and protein constituting the PSyD moreover large number of soluble and membrane bound molecules serving functions in synaptic signal transduction and metabolism were detected the results provide an inventory of the synaptic proteome and confirm the suitability of the ICAT method for the assessment of synaptic structure function and plasticity

biomarkers for cancer risk early detection prognosis and therapeutic response promise to revolutionize cancer management protein biomarkers offer tremendous potential in this regard due to their great diversity and intimate involvement in physiology An effective program to discover protein biomarkers using existing technology will require team science an integrated informatics platform identification and quantitation of candidate biomarkers in disease tissue mouse models of disease standardized reagents for analyzing candidate biomarkers in bodily fluids and implementation of automation technology improvements for better fractionation of the proteome selection of specific biomarkers from complex mixtures and multiplexed assay of biomarkers would greatly enhance progress

technologies for genome wide analyses typically undergo transition from discovery phase to scoring phase In the discovery phase the genomic universe is explored and all pertinent data are noted In the scoring phase relevant entities are screened to reveal groups of gene that are associated with specific biological process or conditions In this article we propose that the transition from discovery to scoring phase is also essential feasible and imminent for proteomics

biomarkers to assist in the diagnosis and medical management of alzheimer disease AD are pressing need We have employed proteomic approach microcapillary liquid chromatography mass spectrometry of protein labeled with isotope coded affinity tags ICAT to quantify relative changes in the proteome of human cerebrospinal fluid CSF obtained from the lumbar cistern using CSF from well characterized AD patients and age matched controls at different institutions we quantified protein concentration ratios of of the CSF protein that we have identified and found differences in over half of them We confirmed our findings by western blot and validated this approach by quantifying relative levels of amyloid precursor protein and cathepsin in AD patients and control individuals quantitative proteomics of CSF from AD patients compared to age matched controls as well as from other neurodegenerative diseases will allow us to generate roster of protein that may serve as specific biomarker panels for AD and other geriatric dementias

the maturation of MS technologies has provided rich opportunity to interrogate protein expression patterns in normal and disease states by applying expression protein profiling method major goals of this research strategy include the identification of protein biomarkers that demarcate normal and disease populations and the identification of therapeutic biomarkers for the treatment of diseases such as cancer celis and gromov proteomics in translational cancer research toward an integrated approach cancer cell prostate cancer is one disease that would greatly benefit from implementing MS based expression profiling method because of the need to stratify the disease based on molecular markers In this review we will summarize the current MS based method to identify and validate biomarkers in human prostate cancer lastly we propose reverse proteomic approach implementing quantitative MS research strategy to identify and quantify biomarkers implicated in prostate cancer development with this approach the absolute levels of prostate cancer biomarkers will be identified and quantified in normal and diseased samples by measuring the levels of native peptide biomarkers in relation to chemically identical but isotopically labeled reference peptide ultimately centralized prostate cancer peptide biomarker expression database could function as repository for the identification quantification and validation of protein biomarker during prostate cancer progression in men

pancreatic cancer is uniformly lethal disease that is difficult to diagnose at early stage and even more difficult to cure In recent years there has been substantial interest in applying proteomics technologies to identify protein biomarkers for early detection of cancer quantitative proteomic profiling of body fluids tissues or other biological samples to identify differentially expressed protein represents very promising approach for improving the outcome of this disease protein associated with pancreatic cancer identified through proteomic profiling technologies could be useful as biomarkers for the early diagnosis therapeutic targets and disease response markers In this article we discuss recent progress and challenges for applying quantitative proteomics technologies for biomarker discovery in pancreatic cancer

proteins often function as components of larger complexes to perform specific function and formation of these complexes maybe regulated for example intracellular signalling events often require transient and or regulated protein protein interactions for propagation and protein binding to specific DNA sequence RNA molecule or metabolite is often regulated to modulate particular cellular function thus characterizing protein complexes can offer important insights into protein function this review describes recent important advances in mass spectrometry MS based techniques for the analysis of protein complexes following brief descriptions of how protein are identified using MS and general protein complex purification approaches we address two of the most important issues in these types of studies specificity and background protein contaminants two basic strategies for increasing specificity and decreasing background are presented whereas tandem affinity purification TAP of tagged protein of interest can dramatically improve the signal to noise ratio via the generation of cleaner samples stable isotopic labelling of protein may be used to discriminate between contaminants and bona fide binding partners using quantitative MS techniques examples as well as advantages and disadvantages of each approach are presented

identification of cerebrospinal fluid CSF biomarkers of the common age related neurodegenerative diseases would be of great value to clinicians because of the difficulties in differential diagnoses of these diseases in clinical practice protein are one class of potential biomarkers currently under investigation in the hope that different ensembles of protein will aid in the diagnosis of these diseases as well as in the assessment of progression and response to therapy however before undertaking rational approach to CSF protein biomarkers of age related neurodegeneration we must first systematically identify CSF protein and determine whether their levels change with normal aging In this study we used powerful shotgun proteomic method two dimensional microcapillary liquid chromatography electrospray ionization tandem mass spectrometry to identify protein in human CSF additionally using pooled CSF samples we quantitatively compared the CSF proteome of younger adults with that of older adults using isotope coded affinity tags ICAT from these studies we identified more than protein in CSF and found that there were protein with change in concentrations between older and younger individuals finally we validated changes in concentration for two of these protein using western blots in CSF from separate set of individuals these data not only expand substantially our current knowledge regarding human CSF protein but also supply the necessary information to appropriately interpret protein biomarkers of age related neurodegenerative diseases

It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this information can be extracted via quantitative proteomic measurements suitable proteomic techniques need to be sensitive reproducible and robust to detect potential biomarkers below the level of highly expressed protein generate data sets that are comparable between experiments and laboratories and have high throughput to support statistical studies here we report method for high throughput quantitative analysis of serum protein It consists of the selective isolation of peptide that are linked glycosylated in the intact protein the analysis of these now deglycosylated peptide by liquid chromatography electrospray ionization mass spectrometry and the comparative analysis of the resulting patterns By focusing selectively on few formerly linked glycopeptides per serum protein the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptide from unfractionated samples We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns We further identify by tandem mass spectrometry some of the peptide that were consistently elevated in cancer mice compared with their control littermates

quantitative protein profiling using the isotope coded affinity tag ICAT method and tandem mass spectrometry MS enables the pair wise comparison of protein expression levels in biological samples new version of the ICAT reagent with an acid cleavable bond which allows removal of the biotin moiety prior to MS and which utilizes 13C substitution for 12C in the heavy ICAT reagent rather than 2H for 1H as in the original reagent gygi rist gerber frantisek gelb aebersold quantitative analysis of complex protein mixtures using isotope coded affinity tags nat biotechnol was investigated We developed and validated an MS data acquisition strategy using this new reagent that results in an increased number of protein identifications per experiment without losing the accuracy of protein quantification this was achieved by following single survey precursor ion scan and serial collision induced dissociations CIDs of four different precursor ions observed in the prior survey scan this strategy is common to many high performance liquid chromatography electrospray ionization HPLC ESI MS shotgun proteomic strategies but heretofore not to ICAT experiments this advance is possible because the new ICAT reagent uses 13C as the heavy element rather than 2H thus eliminating the slight delay in retention time of ICAT labeled light peptide on C18 based HPLC separation that occurs with 2H and 1H analyses using this new scheme of an ICAT labeled trypsin digested six protein mixture as well as tryptic digest of total yeast lysate indicated that about two times more protein were identified in single analysis and that there was no loss in accuracy of quantification

mass spectrometry based quantitative proteomics has become an important component of biological and clinical research current method while highly developed and powerful are failing short of their goal of routinely analyzing whole proteomes mainly because the wealth of proteomic information accumulated from prior studies is not used for the planning or interpretation of present experiments the consequence of this situation is that in every proteomic experiment the proteome is rediscovered In this report we describe an approach for quantitative proteomics that builds on the extensive prior knowledge of proteomes and platform for the implementation of the method the method is based on the selection and chemical synthesis of isotopically labeled reference peptide that uniquely identify particular protein and the addition of panel of such peptide to the sample mixture consisting of tryptic peptide from the proteome in question the platform consists of peptide separation module for the generation of ordered peptide arrays from the combined peptide sample on the sample plate of MALDI mass spectrometer high throughput MALDI TOF TOF mass spectrometer and suite of software tools for the selective analysis of the targeted peptide and the interpretation of the results applying the method to the analysis of the human blood serum proteome we demonstrate the feasibility of using mass spectrometry based proteomics as high throughput screening technology for the detection and quantification of targeted protein in complex system

protein glycosylation is critically important to biological function and disease state An effective method for the selective isolation of linked glycopeptides has been developed based on the conjugation of glycoproteins to solid support using hydrazine chemistry due to extreme sample complexity separation method such as nano LC MS has been used as an analytical tool for human serum samples but we illustrate here that microfluidic device can achieve superior performance in both sensitivity and reproducibility for profiling complex samples such as glycosylated protein copyright

We have combined classical subcellular fractionation with large scale quantitative mass spectrometry to identify protein that enrich specifically with peroxisomes of saccharomyces cerevisiae In two complementary experiments isotope coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of protein during the purification of peroxisomes mathematical modeling of the data from quantified protein led to prioritized list of candidates whose enrichment scores indicated high likelihood of them being peroxisomal among these protein eight novel peroxisome associated protein were identified the top novel peroxisomal candidate was the small GTpase rho1p although rho1p has been shown to be tethered to membranes of the secretory pathway we show that it is specifically recruited to peroxisomes upon their induction in process dependent on its interaction with the peroxisome membrane protein pex25p rho1p regulates the assembly state of actin on the peroxisome membrane thereby controlling peroxisome membrane dynamics and biogenesis

copper ions switch the oxidation of methane by soluble methane monooxygenase to particulate methane monooxygenase in methylococcus capsulatus bath toward understanding the change in cellular metabolism related to this transcriptional and metabolic switch we have undertaken genomic sequencing and quantitative comparative analysis of the proteome in capsulatus bath grown under different copper to biomass ratios by cleavable isotope coded affinity tag technology Of the protein identified the expressions of protein were stimulated by at least fold by copper ions protein were down regulated by fold or more the protein overexpressed included the methane and carbohydrate metabolic enzymes while the protein suppressed were mainly responsible for cellular signaling process indicating role of copper ions in the expression of the gene associated with the metabolism of the organism downstream of methane oxidation the study has also provided complete map of the C1 metabolism pathways in this methanotroph and clarified the interrelationships between them

expression of the CDK inhibitor p21cip1 is tightly regulated by signals that control cell division p21 is an unstable protein that is degraded by the proteasome however the pathway that leads to proteasomal degradation of p21 has proven to be enigmatic An important issue is whether proteasomal degradation of p21 occurs independently of ubiquitylation or alternatively whether ubiquitylation on its terminus is crucial We resolve this uncertainty by showing that endogenous cellular p21 is completely acetylated at its amino terminus and is therefore not substrate for ubiquitylation We further show that inactivation of essential components of the ubiquitylation machinery does not directly impact endogenous p21 degradation our results underscore the importance of acetylation in restricting ubiquitylation and show in particular that ubiquitylation of endogenous p21 either at internal lysines or on the terminus is unlikely to control its degradation by the proteasome

BACKGROUND interferons IFNs play critical role in the host antiviral defense and are an essential component of current therapies against hepatitis virus HCV major cause of liver disease worldwide To examine liver specific responses to IFN and begin to elucidate the mechanisms of IFN inhibition of virus replication we performed global quantitative proteomic analysis in human hepatoma cell line huh7 in the presence and absence of IFN treatment using the isotope coded affinity tag ICAT method and tandem mass spectrometry MS MS RESULTS In three subcellular fractions from the huh7 cell treated with IFN IU ml or mock treated we identified more than protein at threshold that corresponds to less than false positive error rate among these were induced by IFN and were repressed by more than two fold respectively these IFN regulated protein represented multiple cellular functions including antiviral defense immune response cell metabolism signal transduction cell growth and cellular organization To analyze this proteomics dataset we utilized several system biology data mining tools including gene ontology via the Gominer program and the cytoscape bioinformatics platform CONCLUSIONS integration of the quantitative proteomics with global protein interaction data using the cytoscape platform led to the identification of several novel and liver specific key regulatory components of the IFN response which may be important in regulating the interplay between HCV interferon and the host response to virus infection

abnormal centrosomal structures similar to those occurring in human cancers are induced in fission yeast by overexpression of the pericentrin homolog pcp1p analysis of abnormal pcp1p containing structures with quantitative mass spectrometry and isotope coded affinity tags identified coiled coil structural maintenance of chromosomes SMC domain protein this protein termed ccq1p coiled coil protein quantitatively enriched localizes with taz1p to telomeres in normal vegetative cell fluorescence resonance energy transfer FRET measurements indicate that ccq1p also interacts with centrosomal pcp1p in mating pheromone stimulated cell containing centrosomally clustered telomeres We provide evidence that the ccq1p pcp1p interaction while essential for meiosis is deleterious when forced to occur during vegetative growth cell lacking one ccq1 allele exhibit loss of function phenotype including abnormally long cell length chromosome segregation failure telomeric shortening and defective telomeric clustering during meiotic prophase our data indicate mechanism underlying meiotic chromosomal bouquet formation and suggest recruitment model for supernumerary centrosome toxicity

this review focuses on how membrane lipid rafts have been detected and isolated mostly from lymphocytes and their associated protein identified these protein include transmembrane antigens receptors GPI anchored protein cytoskeletal protein src family protein kinases protein and other protein involved in signal transduction To further understand the biology of lipid rafts new methodological approaches are needed to help characterize the raft protein component and changes that occur in this component as result of cell perturbation We describe the application of new proteomic approaches to the identification and quantification of raft protein in lymphocytes similar approaches applied to other model cell system will provide valuable new insights into both cellular signal transduction and lipid raft biology

A broad range of mass spectrometers are used in mass spectrometry MS based proteomics research each type of instrument possesses unique design data system and performance specifications resulting in strengths and weaknesses for different types of experiments unfortunately the native binary data formats produced by each type of mass spectrometer also differ and are usually proprietary the diverse nontransparent nature of the data structure complicates the integration of new instruments into preexisting infrastructure impedes the analysis exchange comparison and publication of results from different experiments and laboratories and prevents the bioinformatics community from accessing data sets required for software development here we introduce the mzXML format an open generic XML extensible markup language representation of MS data We have also developed an accompanying suite of supporting programs We expect that this format will facilitate data management interpretation and dissemination in proteomics research

ß catenin is central effector of wnt signaling in embryonic and stem cell development and in tumorigenesis here through mass spectrometric analysis of catenin protein complex we identified protein as putative catenin interactors We show that one of them enhances catenin dependent transcription by maintaining high level of catenin protein in the cytoplasm more importantly facilitates activation of catenin by the survival kinasa akt and colocalizes with activated akt in intestinal stem cell We propose that akt phosphorylates catenin which results in binding and stabilization of catenin and these interactions may be involved in stem cell development

proteins from human liver carcinoma huh7 cell representing transformed liver cell and cultured primary human fetal hepatocytes HFH and human HH4 hepatocytes representing nontransformed liver cell were extracted and processed for proteome analysis protein from stimulated cell interferon treatment for the huh7 and HFH cell and induction of hepatitis virus HCV protein for the HH4 cell and corresponding control cell were labeled with light and heavy cleavable ICAT reagents respectively the labeled samples were combined trypsinized and subject to cation exchange and avidin affinity chromatographies the resulting cysteine containing peptide were analyzed by microcapillary LC MS MS the MS MS spectra were initially analyzed by searching the human international protein index database using the SEQUEST software subsequently new statistical algorithms were applied to the collective SEQUEST search results of each experiment first the peptideprophet software was applied to discriminate true assignments of MS MS spectra to peptide sequence from false assignments to assign probability value for each identified peptide and to compute the sensitivity and error rate for the assignment of spectra to sequence in each experiment second the proteinprophet software was used to infer the protein identifications and to compute probabilities that protein had been correctly identified based on the available peptide sequence evidence the resulting protein lists were filtered by proteinprophet probability score which corresponded to an error rate of less than total of and protein or related protein groups were identified in three subdatasets from the huh7 HFH and HH4 cell respectively In total these subdatasets contained unique protein identifications from human liver cell An increase of the threshold to corresponding to an error rate of less than resulted in unique protein identifications and for the huh7 HFH and HH4 cell respectively

using DNA microarrays together with quantitative proteomic techniques ICAT reagents two dimensional DIGE and MS we evaluated the correlation of mRNA and protein levels in two hematopoietic cell lines representing distinct stages of myeloid differentiation as well as in the livers of mice treated for different periods of time with three different peroxisome proliferative activated receptor agonists We observe that the differential expression of mRNA up or down can capture at most of the variation of protein expression although the overall pattern of protein expression is similar to that of mRNA expression the incongruent expression between mRNAs and protein emphasize the importance of posttranscriptional regulatory mechanisms in cellular development or perturbation that can be unveiled only through integrated analyses of both protein and mRNAs

To identify the protein associated with soluble synuclein AS that might promote AS aggregation key event leading to neurodegeneration we quantitatively compared protein profiles of AS associated protein complexes in MES cell exposed to rotenone pesticide that produces parkinsonism in animals and induces lewy body LB like inclusions in the remaining dopaminergic neurons and to vehicle We identified more than protein associated with nonidet soluble AS and demonstrated that at least of these protein displayed significant differences in their relative abundance in AS complexes under conditions where rotenone was cytotoxic and induced formation of cytoplasmic inclusions immunoreactive to anti AS overexpressing one of these protein heat shock protein hsp not only protected cell from rotenone mediated cytotoxicity but also decreased soluble AS aggregation furthermore the protection afforded by hsp70 transfection appeared to be related to suppression of rotenone induced oxidative stress as well as mitochondrial and proteasomal dysfunction

recent data in invertebrates demonstrated that huntingtin htt is essential for fast axonal trafficking here we provide direct and functional evidence that htt is involved in fast axonal trafficking in mammals moreover expression of full length mutant htt mhtt impairs vesicular and mitochondrial trafficking in mammalian neurons in vitro and in whole animals in vivo particularly mitochondria become progressively immobilized and stop more frequently in neurons from transgenic animals these defects occurred early in development prior to the onset of measurable neurological or mitochondrial abnormalities consistent with progressive loss of function wild type htt trafficking motors and mitochondrial components were selectively sequestered by mhtt in human huntington disease affected brain data provide model for how loss of htt function causes toxicity mhtt mediated aggregation sequesters htt and components of trafficking machinery leading to loss of mitochondrial motility and eventual mitochondrial dysfunction

here we describe method for stable isotope labeling and solid phase capture of cysteinyl peptide from complex protein mixtures site specific quantitative labeling of cysteine residue with tags that differ in isotopic content enables quantification of relative peptide abundance between samples labeling on solid phase provides for simultaneous simplification of complex peptide mixture by isolating cysteinyl and subsequently tagged peptide peptide from proteolytic digests of protein samples are labeled in preparation for analysis by microcapillary liquid chromatography and tandem mass spectrometry microLC MS MS to determine their sequence and relative abundance between samples this approach enables rapid identification and accurate quantification of relative abundance of individual protein from different biological contexts

the identification of protein in complex mixtures is most usefid when quantitative information is also obtained We describe new type of protein tagging reagent called the visible isotope coded affinity tag VICAT which allows the absolute amount of target protein or protein to be quantified in complex biological sample such as eukaryotic cell lysate VICAT reagents tag thiol groups of cysteines or thioacetylated amino groups and introduce into the tryptic peptide biotin affinity handle visible moiety for tracking the chromatographic location of the target peptide by method other than mass spectrometry photocleavable linker for removing portion of the tag and an isotope tag for distinguishing sample and internal standard peptide We demonstrate the use of VICAT reagents together with isoelectric focusing of peptide on an immobilized gel strip followed by combined microliquid chromatography electrospray ionization mass spectrometry operating in selected reaction monitoring mode to determine the absolute abundance of specific protein human group phospholipase A2 in eukaryotic cell lysates It is found that human lung macrophages contain fmol of this protein per µg of cell protein western blot analysis of human group phospholipase A2 in macrophages gave inconclusive data VICAT reagents should be useful for numerous applications including the analysis of candidate disease markers in complex mixtures such as serum

We present software tool for visualizing data obtained from analyzing complex peptide mixtures by liquid chromatography LC electrospray ionization ESI mass spectrometry MS the data are represented as two dimensional density plot for experiments employing collision induced dissociation CID links are embedded in the image to the CID spectra and the corresponding peptide sequence that are represented by the respective feature the image provides an intuitive method to evaluate sample quality and the performance of an LC ESI MS system and can be used to optimize experimental conditions local patterns of the image can also be used to identify chemical contaminants and specific peptide features therefore this software tool may have broad application in MS based proteomics

We previously described the use of quantitative proteomics to study macromolecular complexes1 applying the method to analyze yeast RNA polymerase II preinitiation complex we identified new kDa protein encoded by the uncharacterized open reading frame YDR079c as potential new component of the preinitiation complex here we show that YDR079c is bona fide component of polymerase II preinitiation complexes and investigate its role in transcription YDR079c is recruited to promoters both in vivo and in vitro and is required for efficient transcription in vitro and for normal induction of GAL gene In addition YDR079c is core component of general transcription and DNA repair factor IIH and is required for efficient recruitment of TFIIH to promoter yeast lacking YDR079c grow slowly and like strains carrying mutations in core TFIIH subunits are sensitive to ultraviolet radiation YDR079c is conserved throughout evolution and mutations in the human ortholog account for DNA repair deficient form of the tricothiodystrophy disorder called TTD A2 the identification of new evolutionarily conserved core TFIIH subunit is essential for our understanding of TFIIH function in transcription DNA repair and human disease

DNA repair deficient trichothiodystrophy TTD results from mutations in the XPD and XPB subunits of the DNA repair and transcription factor TFIIH In third form of DNA repair deficient TTD called group none of the nine subunits encoding TFIIH carried mutations instead the steady state level of the entire complex was severely reduced new tenth TFIIH subunit TFB5 was recently identified in yeast here we describe the identification of the human TFB5 ortholog and its association with human TFIIH microinjection of cDNA encoding TFB5 GTF2H5 also called TTDA corrected the DNA repair defect of TTD cell and we identified three functional inactivating mutations in this gene in three unrelated families with TTD the GTF2H5 gene product has role in regulating the level of TFIIH the identification of new evolutionarily conserved subunit of TFIIH implicated in TTD provides insight into TFIIH function in transcription DNA repair and human disease

recent technological advances in genomics proteomics and bioinformatics have offered new insights into the molecular mechanisms that underlie lymphocyte signaling and function and the development of new tools in these areas has opened up new avenues for biological investigation By adding quantitative dimension to lymphocyte proteome profiling molecular machines and spatiotemporal regulatory process can now be analyzed using such discovery driven approaches biologists employing genomic and proteomic tools are gathering data at increasing speed and their struggle to extract maximal biological information is helped by new software tools that enable the detailed comparison of multiple datasets

We present the first large scale proteomic analysis of human cellular response to pathogen enteropathogenic escherichia coli EPEC is an enteric human pathogen responsible for much childhood morbidity and mortality worldwide EPEC uses type III secretion system TTSS to inject bacterial protein into the cytosol of intestinal epithelial cell resulting in diarrhea We analyzed the host response to TTSS delivered EPEC effector protein by infecting polarized intestinal epithelial monolayers with either wild type or TTSS deficient EPEC host protein were isolated and subjected to quantitative profiling using isotope coded affinity tagging ICAT combined with electrospray ionization tandem mass spectrometry We identified over unique protein from infected caco monolayers of which are expressed differentially in the presence of TTSS delivered EPEC effector protein We validated these data in silico and through immunoblotting and immunofluorescence microscopy the identified changes extend cytoskeletal observations made in less relevant cell types and generate testable hypotheses with regard to host protein potentially involved in EPEC induced diarrhea these data provide framework for future biochemical analyses of host pathogen interactions

the transcriptome provides the database from which cell assembles its collection of protein translation of individual mRNA species into their encoded protein is regulated producing discrepancies between mRNA and protein levels using new modeling approach to data analysis striking diversity is revealed in association of the transcriptome with the translational machinery each mRNA has its own pattern of ribosome loading circumstance that provides an extraordinary dynamic range of regulation above and beyond actual transcript levels using this approach together with quantitative proteomics we explored the immediate changes in gene expression in response to activation of mitogen activated protein kinase pathway in yeast by mating pheromone interestingly in of those transcripts where the predicted protein synthesis rate changed by at least fold more than half of these changes resulted from altered translational efficiencies these observations underscore that analysis of transcript level albeit extremely important is insufficient by itself to describe completely the phenotypes of cell under different conditions

currently the field of shotgun proteomics relies primarily on the separation of peptide by reversed phase microcapillary chromatography RP µLC combined with either electrospray ionization ESI or matrix assisted laser desorption ionization MALDI and tandem mass spectrometry MS MS for protein identification as well as quantification for this purpose we herein describe construction of RP µLC ESI column emitter along with optimized µLC conditions for using the device to quantify pair wise changes in protein expression via the isotope coded affinity tag ICAT method that also maximize peak capacity these optimized RP µLC parameters required balance be reached between the disparate needs of quantification which requires good peak shape and identification proteome coverage of protein via peptide collision induced dissociation CID which requires peak capacity be maximized complex biological sample from study of murine acetaminophen toxicity in hepatocyes was chosen for method development because of the high level complexity but the biological results are not the focus of this manuscript

quantitative real time measurement of kinetics of sequence specific binding of DNA binding protein to double stranded DNA dsDNA immobilized in array on planar gold surface is demonstrated using surface plasmon resonance SPR microscopy this binding of the yeast transcription factor gal4 to spot dsDNA array made with alternating µm spots of its dsDNA operator sequence and an unrelated DNA sequence proves that this method could be used to simultaneously monitor the kinetics of binding of protein to different dsDNA sequence with sensitivity to pg lt molecules of bound protein in each array spot at time resolution of the method is label free and also allows absolute quantitative determination of the binding stoichiometry the number of protein bound per dsDNA at each time the dsDNA array was fabricated using robotic microspotting system to deliver nanoliter droplets of biotinylated dsDNA solutions onto streptavidin linker layer immobilized with biotinylated alkylthiols on thin gold film simultaneous monitoring of binding to the many array elements allows the use of reference spots array elements with unrelated dsDNA sequence to correct the signal for nonspecific protein DNA binding and changes in bulk refractive index of the solutions in the SPR microscope flow cell this allows high throughput analyses of the kinetics and equilibrium of protein dsDNA binding

ctf8p is component of ctf18 RFC an alternative replication factor like complex required for efficient sister chromatid cohesion in saccharomyces cerevisiae We performed synthetic genetic array SGA analysis with ctf8 deletion strain as primary screen to identify other nonessential gene required for efficient sister chromatid cohesion We then assessed proficiency of cohesion at three chromosomal loci in strains containing deletions of the gene identified in the ctf8 SGA screen deletion of seven gene CHL1 CSM3 BIM1 KAR3 TOF1 CTF4 and VIK1 resulted in defective sister chromatid cohesion mass spectrometric analysis of immunoprecipitated complexes identified physical association between kar3p and vik1p and an interaction between csm3p and tof1p that we confirmed by coimmunoprecipitation from cell extracts these data indicate that synthetic genetic array analysis coupled with specific secondary screens can effectively identify protein complexes functionally related to reference gene furthermore we find that gene involved in mitotic spindle integrity and positioning have previously unrecognized role in sister chromatid cohesion

transcriptional regulatory element trex is positive control site within the muscle creatine kinase MCK enhancer cell culture and transgenic studies indicate that the trex site is important for MCK expression in skeletal and cardiac muscle after selectively enriching for the trex binding factor trexBF using magnetic beads coupled to oligonucleotides containing either wild type or mutant trex sites quantitative proteomics was used to identify trexBF as six4 homeodomain transcription factor of the six sine oculis family from background of copurifying protein using gel shift assay and six specific antisera we demonstrated that six4 is trexBF in mouse skeletal myocytes and embryonic day chick skeletal and cardiac muscle while six5 is the major trexBF in adult mouse heart In cotransfection studies six4 transactivates the MCK enhancer as well as muscle specific regulatory regions of aldolase and cardiac troponin via trex MEF3 sites our results are consistent with six4 being key regulator of muscle gene expression in adult skeletal muscle and in developing striated muscle the trex MEF3 composite sequence ACC GA allowed us to identify novel putative six binding sites in six other muscle gene our proteomics strategy will be useful for identifying transcription factors from complex mixtures using only defined DNA fragments for purification

identification of protein in complex mixtures by mass spectrometry is most useful when quantitative data is also obtained We recently introduced isotope coded affinity tags ICAT reagents for the relative quantification of protein present in two or more biological samples In this report we describe new generation of ICAT reagents that contain the following additional features visible tag that allows the electrophoretic position of tagged peptide during separation to be easily monitored photocleavable linker that allows most of the tag to be removed prior to mass spectrometric analysis an isotope tag that contains carbon and nitrogen atoms instead of deuterium to ensure precise comigration of light and heavy tagged peptide by reverse phase HPLC these reagents contain an iodoacetyl group that selectively reacts with peptide cysteine residue peptide modification chemistry is also reported that allows tagging of peptide that are devoid of cysteine the synthesis of these visible isotope coded affinity tags VICAT reagents and their reaction with peptide are described in this report VICAT reagents containing carbon visible probe or an NBD fluorophore are described these reagents are most useful for the determination of the absolute quantity of specific target protein in complex protein mixtures such as serum or cell lysates

protein tyrosine phosphatases PTpases play key roles in regulating tyrosine phosphorylation levels in cell yet the identity of their substrates remains limited We report here on the identification of PTpases capable of dephosphorylating the phosphorylated immune tyrosine based activation motifs present in the cell receptor subunit To characterize these PTpases we purified enzyme activities directed against the phosphorylated cell receptor subunit by combination of anion and cation chromatography procedures novel ELISA based PTpase assay was developed to rapidly screen protein fractions for enzyme activity following the various chromatography steps We present data that SHP and PTPH1 are present in highly enriched protein fractions that exhibit PTpase activities toward tyrosine phosphorylated TCR substrate specific activity ranging from to pmol min µg We also used protein tyrosine phosphatase substrate trapping library comprising the catalytic domains of distinct protein tyrosine phosphatases representing almost all the tyrosine phosphatases identified in the human genome PTPH1 was the predominant phosphatase capable of complexing phospho subsequent transfection assay indicated that SHP and PTPH1 are the two principal PTpases capable of regulating the phosphorylation state of the TCR ITAMs with PTPH1 directly dephosphorylating this is the first reported demonstration that PTPH1 is candidate PTpase capable of interacting with and dephosphorylating TCR

We present two strategies for microspotting arrays of double stranded DNAs dsDNAs onto gold coated glass slide for high throughput studies of protein DNA interactions by surface plasmon resonance SPR microscopy both method use streptavidin SA as linker layer between biotin containing mixed self assembled monolayer SAM and biotinylated dsDNAs to produce arrays with high packing density the primary mixed SAM is produced from biotin and oligo ethylene glycol terminated thiols bonded as thiolates onto the gold surface In the first method robotic microspotter is used to deliver nanoliter droplets of dsDNA solution onto uniform layer of this SA SA cm2 SPR microscopy shows density of dsDNA cm dsDNA SA in the array elements the second method uses instead microspotted array of this SA linker layer onto which the microspots of dsDNA are added with spatial registry SPR microscopy before addition of the dsDNA shows SA coverage of SA cm2 within the spots and dsDNA density of dsDNA cm2 dsDNA SA depending on the length of dsDNA after dsDNA spotting We demonstrate the ability to simultaneously monitor protein binding with the SPR microscope in many µm spots with time resolution and sensitivity to lt pg of protein

tandem mass spectrometry has been used increasingly for high throughput analysis of complex protein samples major challenge lies in the consistent objective and transparent analysis of the large amounts of data generated by such experiments and in their dissemination and publication here we review currently available computational tools and discuss the need for statistical criteria in the analysis of large proteomics datasets

identification and characterization of multi protein complexes is an important step toward an integrative view of protein protein interaction networks that determine protein function and cell behavior the limiting factor for identifying protein complexes is the method for their separation blue native PAGE BN PAGE permits high resolution separation of multi protein complexes under native conditions To date BN PAGE has only been applicable to purified material here we show that dialysis permits the analysis of multi protein complexes of whole cellular lysates by BN PAGE We visualized different multi protein complexes by immunoblotting including forms of the eukaryotic proteasome complex dynamics after interferon stimulation of cell was studied and an antibody shift assay was used to detect protein protein interactions in BN PAGE furthermore we identified defined protein complexes of various protein including the tumor suppressor p53 and myc finally we identified multi protein complexes via mass spectrometry showing that the method has wide potential for functional proteomics

quantitative proteome profiling using mass spectrometry and stable isotope dilution is being widely applied for the functional analysis of biological system and for the detection of clinical diagnostic or prognostic marker protein because of the enormous complexity of proteomes their comprehensive analysis is unlikely to be routinely achieved in the near future however in recent years significant progress has been achieved focusing quantitative proteomic analyses on specific protein classes or subproteomes that are rich in biologically or clinically important information such projects typically combine the use of chemical probes that are specific for targeted group of protein and may contain stable isotope signatures for accurate quantification with automated tandem mass spectrometry and bioinformatics tools for data analysis In this review we summarize technical and conceptual advances in quantitative subproteome profiling based on tandem mass spectrometry and chemical probes

prostate cancer is unusual among neoplasms in that it may be diagnosed at curable stage through detection of protein in serum the serine protease prostate specific antigen PSA PSA is secreted by both normal and neoplastic prostate epithelial cell in response to androgenic hormones and has found widespread use in cancer screening because PSA screening is controversial due to sensitivity and specificity issues efforts continue to focus on the identification and characterization of additional markers that may be used for diagnostic and therapeutic purposes In this study we report the application of quantitative proteomic techniques that incorporate isotope coded affinity tag reagents and tandem mass spectrometry to comprehensively identify secreted and cell surface protein from neoplastic prostate epithelium LNCaP cell prostate tumor derived cell line that secretes PSA in response to androgen exposure were grown in low protein defined media under androgen stimulated A+ and starved conditions proteomic analysis of the media identified in excess of protein of which could be quantified nine percent of the protein had A+ ratios including PSA and had ratios subset of these androgen regulated protein appeared to be expressed in abundance Of these selected mass spectrometry observations were confirmed by western analysis the findings suggest that androgen mediated release of protein may occur through the activation of proteolytic enzymes rather than exclusively through transcriptional or translational control mechanisms On the basis of their known functional roles several of the abundant androgen regulated protein may participate in the progression of neoplastic epithelial cell growth and should be considered as potential serum markers of neoplastic prostate diseases

classical proteomics combined two dimensional gel electrophoresis DE for the separation and quantification of protein in complex mixture with mass spectrometric identification of selected protein more recently the combination of liquid chromatography LC stable isotope tagging and tandem mass spectrometry MS MS has emerged as an alternative quantitative proteomics technology We have analyzed the proteome of mycobacterium tuberculosis major human pathogen comprising about gene by DE and mass spectrometry MS and by ii the isotope coded affinity tag ICAT reagent method and MS MS the data obtained by either technology were compared with respect to their selectivity for certain protein types and classes and with respect to the accuracy of quantification initial datasets of peptide MS MS spectra and spots for the ICAT LC MS and DE MS method respectively were reduced to and conclusively identified and quantified protein respectively ICAT LC MS showed clear bias for high protein and was complemented by the DE MS method which showed preference for low protein and also identified cysteine free protein that were transparent to the ICAT LC MS method relative quantification between two strains of the tuberculosis complex also revealed that the two technologies provide complementary quantitative information whereas the ICAT LC MS method quantifies the sum of the protein species of one gene product the DE MS method quantifies at the level of resolved protein species including post translationally modified and processed polypeptides our data indicate that different proteomic technologies applied to the same sample provide complementary types of information that contribute to more complete understanding of the biological system studied

during erythroid differentiation globin gene expression is regulated by the locus control region LCR the transcription factor NF E2p18 mafK binds within this region and is essential for globin expression in murine erythroleukemia MEL cell here we use the isotope coded affinity tag ICAT technique of quantitative mass spectrometry to compare protein interacting with NF E2p18 mafK during differentiation our results define mafK as dual function molecule that shifts from repressive to an activating mode during erythroid differentiation the exchange of mafK dimerization partner from bach1 to NF E2p45 is key step in the switch from the repressed to the active state this shift is associated with changes in the interaction of mafK with corepressors and co activators thus our results suggest that in addition to its role as cis acting activator of globin gene expression in differentiated erythroid cell the LCR also promotes an active repression of globin transcription in committed cell before terminal differentiation

critical events for vasoconstrictor and growth factor signal transduction include stimulation of phospholipase PLC and elevation of intracellular calcium src has been proposed as common mediator for these signals activated by both protein coupled receptors GPCRs and tyrosine kinase coupled receptors TKRs here we show that the GPCR kinase interacting protein GIT1 is substrate for src that undergoes tyrosine phosphorylation in response to angiotensin II angII and EGF in vascular smooth muscle and cell GIT1 associates with PLC via the PLC src homology and domains constitutively and the interaction is unaltered by angII and EGF GIT1 interaction with PLC is required for PLC activation based on inhibition of tyrosine phosphorylation and calcium mobilization after GIT1 knockdown with antisense GIT1 oligonucleotides GIT1 interacts with PLC via novel spa homology domain SHD and coiled coil domain deletion mutation analysis showed that GIT1 SHD is required for angII and EGF mediated PLC activation measured by phosphorylation of tyr and inositol trisphosphate formation We propose that GIT1 is novel regulator of PLC function that mediates PLC activation by src and integrates signal transduction by GPCRs and TKRs

BACKGROUND androgens play critical role in the development of prostate cancer dysregulation of androgen regulated growth pathways can led to hormone refractory prostate cancer comprehensive understanding of androgen regulated cellular process has not been achieved to date To this end we have applied large scale proteomic approach to define cellular process that are responsive to androgen treatment in LNCaP prostate cancer cell RESULTS using isotope coded affinity tags and mass spectrometry we identified and quantified the relative abundance levels of protein and found that distinct cellular process were coregulated by androgen while others were essentially unaffected subsequent pharmacological perturbation of the cellular process for energy generation confirmed that androgen starvation had profound effect on this pathway CONCLUSIONS our results provide evidence for the role of androgenic hormones in coordinating the expression of critical components involved in distinct cellular process and further establish foundation for the comprehensive reconstruction of androgen regulated protein networks and pathways in prostate cancer cell

drugs affecting the cell cycle provide insights into mechanisms underlying cancer and suggest strategies for ablating uncontrolled growth essential to an understanding of the activity of such compounds is the identification of the set of protein affected either directly or indirectly by the drug the combination of novel technologies for stable isotope protein tagging chromatographic separation tandem mass spectrometry and data processing is an extremely powerful means for providing such identifications and in addition for establishing proteome wide profile of all protein whose abundance levels or phosphorylation state are affected by the drug

We describe an algorithm for the automated statistical analysis of protein abundance ratios ASAPratio of protein contained in two samples protein are labeled with distinct stable isotope tags and fragmented and the tagged peptide fragments are separated by liquid chromatography LC and analyzed by electrospray ionization ESI tandem mass spectrometry MS MS the algorithm utilizes the signals recorded for the different isotopic forms of peptide of identical sequence and numerical and statistical method such as savitzky golay smoothing filters statistics for weighted samples and dixon test for outliers to evaluate protein abundance ratios and their associated errors the algorithm also provides statistical assessment to distinguish protein of significant abundance changes from population of protein of unchanged abundance To evaluate its performance two sets of LC ESI MS MS data were analyzed by the ASAPratio algorithm without human intervention and the data were related to the expected and manually validated values the utility of the ASAPratio program was clearly demonstrated by its speed and the accuracy of the generated protein abundance ratios and by its capability to identify specific core components of the RNA polymerase II transcription complex within high background of copurifying protein

reversed phase microcapillary chromatography RP µgLC combined with electrospray ionization tandem mass spectrometry ESI MS MS is one of two prevailing techniques in proteomic analysis the other being matrix assisted laser desorption ionization MALDI despite the arguably better dynamic range obtainable with ESI MALDI is increasingly popular due to ease of use ruggedness and the ability to decouple separation from ionization By contrast in order to take advantage of the sensitivity and dynamic range afforded by the concentration dependent nature of ESI it is directly coupled to separations that take place in small RP µLC columns this gain in sensitivity often comes at loss of ruggedness due to clogging of the small RP µLC columns one result of which is limited sample throughput here we describe combined µpre column µLC ESI device that is sensitive rugged and modular in design allowing facile construction and troubleshooting due to low signal to noise as little as attomole of peptide can be selected by data dependent method for collision induced dissociation importantly the resulting tandem mass spectrum is of high enough quality to identify the peptide sequence by database search against complex database using SEQUEST finally the device is demonstrated to be rugged as judged by consecutive reversed phase µLC separations on complex peptide mixtures before chromatographic resolution is degraded copyright

A statistical model is presented for computing probabilities that protein are present in sample on the basis of peptide assigned to tandem mass MS MS spectra acquired from proteolytic digest of the sample peptide that correspond to more than single protein in the sequence database are apportioned among all corresponding protein and minimal protein list sufficient to account for the observed peptide assignments is derived using the expectation maximization algorithm using peptide assignments to spectra generated from sample of purified protein as well as complex influenzae and halobacterium samples the model is shown to produce probabilities that are accurate and have high power to discriminate correct from incorrect protein identifications this method allows filtering of large scale proteomics data sets with predictable sensitivity and false positive identification error rates fast consistent and transparent it provides standard for publishing large scale protein identification data sets in the literature and for comparing the results obtained from different experiments

androgen ablation therapy is an effective method for treating prostate cancer however prostate tumors that survive long term androgen ablation therapy are classified as androgen independent as they proliferate in the absence of androgens and they tend to be enriched for neuroendocrine NE cell androgen withdrawal causes androgen dependent prostate cancer cell to adopt pronounced NE phenotype suggesting that androgen receptor AR represses an intrinsic NE transdifferentiation process in prostate cancer cell In this report we show that short interfering RNA induced AR silencing induced NE phenotype that manifested itself in the growth of dendritic like process in both the androgen dependent LNCaP and androgen independent LNCaP AI human prostate cancer cell western blot analysis revealed that neuronal specific enolase marker of the neuronal lineage was increased by AR knockdown in LNCaP cell the expression levels of the neuronal specific cytoskeletal protein tubulin III nestin and glial acidic fibrillary protein were also characterized in AR knockdown cell most interestingly AR silencing induced tubulin III expression in LNCaP cell while AR knockdown increased glial acidic fibrillary protein levels in both LNCaP and LNCaP AI cell lastly AR silencing reduced the proliferative capacity of LNCaP and LNCaP AI cell our data demonstrate that AR actively represses an intrinsic NE transdifferentiation process in androgen responsive prostate cancer cell and suggest potential link between AR inactivation and the increased frequency of NE cell in androgen independent tumors

halobacterium sp NRC insoluble membrane and soluble cytoplasmic protein were isolated by ultracentrifugation of whole cell lysate using an ion trap mass spectrometer equipped with C18 trap electrospray ionization emitter micro liquid chromatography column number of trypsin generated peptide tags from unique protein were identified this represents approximately one fifth of the theoretical proteome of halobacterium Of these protein were found only in the soluble fraction were only in the insoluble membrane fraction and were in both fractions there were and previously annotated protein identified in the soluble and membrane protein fractions respectively interestingly of previously unannotated protein found only in halobacterium NRC were identified such protein could be interesting targets for understanding unique physiology of halobacterium NRC group of protein involved in various metabolic pathways were identified among the expressed protein suggesting these pathways were active at the time the cell were collected this data containing list of expressed protein their cellular locations and biological functions could be used in future studies to investigate the interaction of the gene and protein in relation to genetic or environmental perturbations

In the previous paper in this journal we reported the use of capillary sieving electrophoresis to characterize protein expressed by single cancer cell at specific phases in the cell cycle analysis of the data revealed one component with cell cycle dependent changes in expression at the confidence limit however the amount of protein present in single cell is far too small to allow its direct identification by mass spectrometry In this paper we report method by which such protein can be tentatively identified We perform standard SDS PAGE electrophoresis of the protein contained within homogenate prepared from an HT29 cell culture protein extracted from bands in the gel are identified by mass spectrometry the protein also provide set of standards that can be used to spike the sample before capillary sieving electrophoresis CSE separation comigration is taken as evidence for the identity of the target protein In proof of principle experiment single band migrating at kDa was isolated from the SDS PAGE gel generated from the HT29 cell line protein extracted from this band were used to spike CSE separation of the same extract this band comigrated with cell cycle dependent component identified from single cell analysis In gel digestion and LC MS MS were used to identify five protein including cytokeratin which is the product of the most highly expressed gene in this cell line

In the previous paper in this journal we reported the use of capillary sieving electrophoresis to characterize protein expressed by single cancer cell at specific phases in the cell cycle analysis of the data revealed one component with cell cycle dependent changes in expression at the confidence limit however the amount of protein present in single cell is far too small to allow its direct identification by mass spectrometry In this paper we report method by which such protein can be tentatively identified We perform standard SDS PAGE electrophoresis of the protein contained within homogenate prepared from an HT29 cell culture protein extracted from bands in the gel are identified by mass spectrometry the protein also provide set of standards that can be used to spike the sample before capillary sieving electrophoresis CSE separation comigration is taken as evidence for the identity of the target protein In proof of principle experiment single band migrating at approximately kDa was isolated from the SDS PAGE gel generated from the HT29 cell line protein extracted from this band were used to spike CSE separation of the same extract this band comigrated with cell cycle dependent component identified from single cell analysis In gel digestion and LC MS MS were used to identify five protein including cytokeratin which is the product of the most highly expressed gene in this cell line

A method to identify and quantify chromatin associated protein has been developed and applied to the analysis of changes in chromatin associated protein induced by myc oncoprotein expression in human lymphocytes chromatin enriched fractions were isolated by differential detergent salt extraction and analyzed by ICAT reagent labeling multi dimensional chromatography and tandem mass spectrometry many known chromatin associated regulatory factors were identified and quantified the method will be widely applicable to various biological system and reveal changes in chromatin associated regulatory factors that underlie biological phenomena

lipid rafts were prepared according to standard protocols from jurkat cell stimulated via cell receptor CD28 cross linking and from control unstimulated cell Co isolating protein from the control and stimulated cell preparations were labeled with isotopically normal d0 and heavy d8 versions of the same isotope coded affinity tag ICAT reagent respectively samples were combined proteolyzed and resultant peptide fractionated via cation exchange chromatography cysteine containing ICAT labeled peptide were recovered via the biotin tag component of the ICAT reagents by avidin affinity chromatography On line micro capillary liquid chromatography tandem mass spectrometry was performed on both avidin affinity ICAT labeled and flow through unlabeled fractions initial peptide sequence identification was by searching recorded tandem mass spectrometry spectra against human sequence data base using SEQUEST software new statistical data modeling algorithms were then applied to the SEQUEST search results these allowed for discrimination between likely correct and incorrect peptide assignments and from these the inferred protein that they collectively represented by calculating estimated probabilities that each peptide assignment and subsequent protein identification was member of the correct population for convenience the resultant lists of peptide sequence assigned and the protein to which they corresponded were filtered at an arbitrarily set cut off of likely to be correct and above and compiled into two separate datasets In total these data sets contained individual peptide identifications which represented unique peptide sequence corresponding to protein and related protein groups

proteomic approaches to biological research that will prove the most useful and productive require robust sensitive and reproducible technologies for both the qualitative and quantitative analysis of complex protein mixtures here we applied the isotope coded affinity tag ICAT approach to quantitative protein profiling in this case protein that copurified with lipid raft plasma membrane domains isolated from control and stimulated jurkat human cell with the ICAT approach cysteine residue of the two related protein isolates were covalently labeled with isotopically normal and heavy versions of the same reagent respectively following proteolytic cleavage of combined labeled protein peptide were fractionated by multidimensional chromatography and subsequently analyzed via automated tandem mass spectrometry individual tandem mass spectrometry spectra were searched against human sequence database and variety of recently developed publicly available software applications were used to sort filter analyze and compare the results of two repetitions of the same experiment In particular robust statistical modeling algorithms were used to assign measures of confidence to both peptide sequence and the protein from which they were likely derived identified via the database searches We show that by applying such statistical tools to the identification of cell lipid raft associated protein we were able to estimate the accuracy of peptide and protein identifications made these tools also allow for determination of the false positive rate as function of user defined data filtering parameters thus giving the user significant control over and information about the final output of large scale proteomic experiments with the ability to assign probabilities to all identifications the need for manual verification of results is substantially reduced thus making the rapid evaluation of large proteomic datasets possible finally by repeating the experiment information relating to the general reproducibility and validity of this approach to large scale proteomic analyses was also obtained

It is frequently debated question whether technology drives biology or whether biology drives the development of new technologies this issue is discussed in this manuscript as an account that covers approximately decade during which mass spectrometry and protein biochemistry have intersected It is shown that the capabilities of the mass spectrometric method initially developed to address the specific need to identify protein reliably and at high sensitivity soon transcended the intended task the rapid development of mass spectrometric technologies applied to protein research has catalyzed entirely new experimental approaches and opened up new types of biological questions to experimentation culminating in the field of proteomics some conclusions from this case study relating to technological research and the environment in which it is carried out are also discussed

In this study large scale qualitative and quantitative proteomic technology was applied to the analysis of the opportunistic bacterial pathogen pseudomonas aeruginosa grown under magnesium limitation an environmental condition previously shown to induce expression of various virulence factors for quantitative analysis whole cell and membrane protein were differentially labeled with isotope coded affinity tag ICAT reagents and ICAT reagent labeled peptide were separated by two dimensional chromatography prior to analysis by electrospray ionization tandem mass spectrometry ESI MS MS in an ion trap mass spectrometer ITMS To increase the number of protein identifications gas phase fractionation GPF in the dimension was employed for analysis of ICAT peptide derived from whole cell extracts the experiments confirmed expression of aeruginosa protein of which were differentially expressed upon limitation of magnesium number of conserved gram negative magnesium stress response protein involved in bacterial virulence were among the most abundant protein induced in low magnesium comparative ICAT analysis of membrane versus whole cell protein indicated that growth of aeruginosa in low magnesium resulted in altered subcellular compartmentalization of large enzyme complexes such as ribosomes this result was confirmed by PAGE analysis of aeruginosa outer membrane protein this study shows that large scale quantitative proteomic technology can be successfully applied to the analysis of whole bacteria and to the discovery of functionally relevant biologic phenotypes

with the completion of the genomic sequencing of number of species including that of humans much attention is currently focused on how the information in these sequence might be interpreted in terms of the structure function and control of biologic system and process quantitative proteome analysis the global analysis of protein expression is increasingly being used as method to study steady state and perturbation induced changes in protein profiles the rationale for quantitative proteome analysis is described along with new technology for high throughput quantitative profiling of protein in complex mixtures and its current status with selected applications

quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry MS MS is an emerging technology with great potential for the functional analysis of biological system and for the detection of clinical diagnostic or prognostic marker protein owing to the enormous complexity of proteomes their comprehensive analysis is an as yet unresolved technical challenge however biologically or clinically important information can be obtained if specific information rich protein classes or sub proteomes are isolated and analyzed glycosylation is the most common post translational modification here we describe method for the selective isolation identification and quantification of peptide that contain linked carbohydrates It is based on the conjugation of glycoproteins to solid support using hydrazide chemistry stable isotope labeling of glycopeptides and the specific release of formerly linked glycosylated peptide via peptide glycosidase PNgase the recovered peptide are then identified and quantified by MS MS We applied the approach to the analysis of plasma membrane protein and protein contained in human blood serum

bim is pro apoptotic member of the bcl protein family bim has three isoforms EL and of which the EL form is the least cytotoxic We show here that bim is serine phosphorylated in lymphocytes predominantly on the EL form withdrawal of IL from IL dependent lymphocytes or culture of thymocytes leads to reduced bim phosphorylation and apoptosis induction this decrease in bim phosphorylation occurs when most cell in culture are still viable indicating that reduction of bim phosphorylation may be an early event in apoptosis signaling of lymphocytes

topoisomerases alter DNA topology and are vital for the maintenance of genomic integrity topoisomerases and II are also targets for widely used antitumor agents We demonstrated previously that in the human leukemia cell line HL resistance to topoisomerase topo II targeting drugs such as etoposide is associated with site specific hypophosphorylation of topo IIa this effect can be mimicked in sensitive cell treated with the intracellular Ca2+ chelator bis aminophenoxy ethane tetraacetic acid BAPTA AM here we identify ser as major phosphorylation site in the catalytic domain of topo IIa this site lies within the consensus sequence for the acidotrophic kinases casein kinase and casein kinase II mutation of serine to alanine S1106A abrogates phosphorylation of phosphopeptides that were found to be hypophosphorylated in resistant HL cell or sensitive cell treated with BAPTA AM purified topo IIa containing S1106A substitution is fold less active than wild type topo IIa in decatenating kinetoplast DNA and also exhibits fold decrease in the level of etoposide stabilized DNA cleavable complex formation saccharomyces cerevisiae JN394t2 cell expressing S1106A mutant topo IIa protein are more resistant to the cytotoxic effects of etoposide or amsacrine these results demonstrate that Ca2+ regulated phosphorylation of ser in the catalytic domain of topo IIa modulates the enzymatic activity of this protein and sensitivity to topo II targeting drugs

yeast TGN resident protein that frequently cycle between the TGN and endosomes are much more slowly transported to the prevacuolar late endosomal compartment PVC than other protein however TGN protein transport to the PVC is accelerated in mutants lacking function of inp53p inp53p contains sacI polyphosphoinositide phosphatase domain phosphatase domain and proline rich domain here we show that all three domains are required to mediate slow delivery of TGN protein into the PVC although deletion of the proline rich domain did not affect general membrane association it caused localization to become less specific the proline rich domain was shown to bind to two protein including clathrin heavy chain chc1p unlike chc1 mutants inp53 mutants do not mislocalize TGN protein to the cell surface consistent with the idea that chc1p and inp53p act at common vesicular trafficking step but that chc1p is used at other steps also like mutations in the AP adaptor complex mutations in INP53 exhibit synthetic growth and transport defects when combined with mutations in the GGA protein taken together with other recent studies our results suggest that inp53p and AP clathrin act together in TGN to early endosome pathway distinct from the direct TGN to PVC pathway mediated by GGA clathrin

recent successes illustrate the role of mass spectrometry based proteomics as an indispensable tool for molecular and cellular biology and for the emerging field of system biology these include the study of protein protein interactions via affinity based isolations on small and proteome wide scale the mapping of numerous organelles the concurrent description of the malaria parasite genome and proteome and the generation of quantitative protein profiles from diverse species the ability of mass spectrometry to identify and increasingly to precisely quantify thousands of protein from complex samples can be expected to impact broadly on biology and medicine

the opportunistic bacterial pathogen pseudomonas aeruginosa colonizes airways of individuals with cystic fibrosis CF with resultant chronic destructive lung disease aeruginosa adaptation to the CF airway includes biofilm formation and antibiotic resistance isolates from asymptomatic individuals in the first years of life have unique characteristics suggesting that adaptation occurs before clinical symptoms one defined early adaptation is expression of specific proinflammatory lipopolysaccharide LPS that is associated with antimicrobial peptide resistance this CF specific LPS is induced when aeruginosa is grown in medium that is limited for magnesium therefore qualitative and quantitative proteomic approaches were used to define aeruginosa protein of which were differentially expressed on limitation of magnesium among protein induced by low magnesium were enzymes essential for production of heptyl hydroxy quinolone the pseudomonas quinolone signal PQS which interacts with the homoserine lactone signaling pathway measurement of PQS in aeruginosa isolates from asymptomatic children with CF indicated that strains with increased synthesis of PQS are present during early colonization of CF patient airways

proteomics is the systematic study of the many and diverse properties of protein in parallel manner with the aim of providing detailed descriptions of the structure function and control of biological system in health and disease advances in method and technologies have catalyzed an expansion of the scope of biological studies from the reductionist biochemical analysis of single protein to proteome wide measurements proteomics and other complementary analysis method are essential components of the emerging system biology approach that seeks to comprehensively describe biological system through integration of diverse types of data and in the future to ultimately allow computational simulations of complex biological system

We describe generic strategy for determining the specific composition changes in the composition and changes in the abundance of protein complexes It is based on the use of isotope coded affinity tag ICAT reagents1 and mass spectrometry to compare the relative abundances of tryptic peptide derived from suitable pairs of purified or partially purified protein complexes In first application the genuine protein components of large RNA polymerase II pol II preinitiation complex PIC were distinguished from background of co purifying protein by comparing the relative abundances of peptide derived from control sample and the specific complex that was purified from nuclear extracts by single step promoter DNA affinity procedure2 In second application peptide derived from immunopurified STE12 protein complexes isolated from yeast cell in different states were used to detect quantitative changes in the abundance of the complexes and to detect dynamic changes in the composition of the samples the use of quantitative mass spectrometry to guide identification of specific complex components in partially purified samples and to detect quantitative changes in the abundance and composition of protein complexes provides the researcher with powerful new tools for the comprehensive analysis of macromolecular complexes

both the generation and the analysis of proteome data are becoming increasingly widespread and the field of proteomics is moving incrementally toward high throughput approaches techniques are also increasing in complexity as the relevant technologies evolve standard representation of both the method used and the data generated in proteomics experiments analogous to that of the MIAME minimum information about microarray experiment guidelines for transcriptomics and the associated MAGE microarray gene expression object model and XML extensible markup language implementation has yet to emerge this hinders the handling exchange and dissemination of proteomics data here we present UML unified modeling language approach to proteomics experimental data describe XML and SQL structured query language implementations of that model and discuss capture storage and dissemination strategies these make explicit what data might be most usefully captured about proteomics experiments and provide complementary routes toward the implementation of proteome repository

the goal of quantitative proteomics is to determine the identity and relative quantity of each protein present in two or more complex protein samples here we describe novel approach to quantitative proteomics It is based on highly accurate algorithm for the automated quantification of chromatographically fractionated isotope coded affinity tagged peptide and MALDI quadrupole time of flight tandem mass spectrometry for their identification the method is capable of detecting and selectively identifying those protein within complex mixture that show difference in relative abundance We demonstrate the effectiveness and the versatility of this approach in the analysis of standard protein mixture protein expression profiling in human prostate cancer cell line model and identification of the specific components of the multiprotein transcriptional machinery in cerevisiae

the high throughput identification and accurate quantification of protein are essential components of proteomic strategies for studying cellular functions and process techniques that are largely based on stable isotope protein or peptide labeling and automated tandem mass spectrometry are increasingly being applied in quantitative proteomic studies over the past year significant progress has been made toward improving and diversifying these technologies with respect to the method for stable isotope labeling process automation and data processing and analysis advances in stable isotope protein labeling and recent biological studies that used stable isotope based quantitative proteomics techniques are reviewed

the high throughput reversed phase microcapillary liquid chromatographic RP µLC system as nanoscale electrospray ionization source for tandem mass spectrometry ESI MS MS was discussed RP µLC serves as nanoscale nano ESI source and improves the sensitivity of the analysis with its capabilities of on line concentration sample cleanup and separation the RP µLC system consists of micro HPLC pump flow splitter an autosampler precolumn C18 sample trap an integrated microcapillary C18 column ESI emitter the entire procedure including injection valve switching sample cleanup separation and MS MS analysis was fully automated and controlled by PC

quantitatie mass spectrometry and immunoprecipitation were used to identify and measure relative abundance of cell receptor TCR complex components It was observed that TCR subunits were identified in the sample that was speifically precipitated by anti CD3e antibody the ZAP was identified in the fraction of d6 labeled peptide from simulated cell protein other than TCR subunits with ratios for non specific associations were also identified

the development of statistical model to estimate the accuracy of peptide identifications made by MS MS and database search using programs like SEQUEST was discussed the discriminant score and tryptic termini NTT distributions of correctly and incorrectly assigned spectra were empirically derived from each data set as mixture model using the expectation maximization EM algorithm using the EM algorithm the distributions were each updated alternatively with the probabilities until fixed point was achieved the computational results show that using the probabilities computed from the model much higher sensitivity for any given error rate can be achieved as compared to the results of using conventional SEQUEST filtering criteira

A method to increase proteome coverage by gas phase fractionation GPF was presented the reproducibility of data dependent ion selection was examined by mass spectrometry GPF was examined with respect to relative ion intensity It was found that the GPF experiment produced times more protein identifications than triplicate analysis

the use of nano LC MALDI QTOF MS to identify and quantify ICAT labeled protein mixtures was analyzed protein expressed by cell under two different physiological conditions were labeled with ICAT reagent on their crysteinyl residue after washing the labeled peptide were eluted from the avidin column and separated by reversed phase nano LC results showed that the technique has great potential for bypassing the 2D gels and the study of protein expression levels from different cell states

the technique for the identification and relative quantification of protein present in two or more complex protein samples was described the advantages of the given technique include increased efficiency and depth of information in the identification of biologically interesting protein the peptide for showing differences in abundance exceeding user defined threshold are then selected for identification by MS MS analysis It was shown that the approach is useful in profiling studies for the identification of important diagnostic disease markers and the characterization of potential therapeutic targets

A great deal of current biological and clinical research is directed at the interpretation of the information contained in the human genome sequence in terms of the structure function and control of biological system and process proteomics the systematic analysis of protein is becoming critical component in this endeavor because proteomic measurements are carried out directly on protein the catalysts and effectors of essentially all biological functions To detect changes in protein profiles that might provide important diagnostic or functional insights proteomic analyses necessarily have to be quantitative this article summarizes recent technological advances in quantitative proteomics

the extremely halophilic archaeon halobacterium NRC can switch from aerobic energy production energy from organic compounds to anaerobic phototrophy energy from light by induction of purple membrane biogenesis the purple membrane is made up of multiple copies of complex of bacterioopsin bop and retinal called bacteriorhodopsin that functions as light driven proton pump light and redox sensing transcription regulator bat regulates critical gene encoding the biogenesis of the purple membrane To better understand the regulatory network underlying this physiological state we report system approach using global mRNA and protein analyses of four strains of halobacterium sp the wild type NRC and three genetically perturbed strains S9 bat+ purple membrane overproducer and two purple membrane deficient strains SD23 bop knockout and SD20 bat knockout the integrated DNA microarray and proteomic data reveal the coordinated coregulation of several interconnected biochemical pathways for phototrophy isoprenoid synthesis carotenoid synthesis and bacteriorhodopsin assembly In phototrophy the second major biomodule for ATP production arginine fermentation is repressed the primary system level insight provided by this study is that two major energy production pathways in halobacterium sp phototrophy and arginine fermentation are inversely regulated presumably to achieve balance in ATP production under anaerobic conditions

We present statistical model to estimate the accuracy of peptide assignments to tandem mass MS MS spectra made by database search applications such as SEQUEST employing the expectation maximization algorithm the analysis learns to distinguish correct from incorrect database search results computing probabilities that peptide assignments to spectra are correct based upon database search scores and the number of tryptic termini of peptide using SEQUEST search results for spectra generated from sample of known protein components we demonstrate that the computed probabilities are accurate and have high power to discriminate between correctly and incorrectly assigned peptide this analysis makes it possible to filter large volumes of MS MS database search results with predictable false identification error rates and can serve as common standard by which the results of different research groups are compared

with the recent quick expansion of DNA and protein sequence databases intensive efforts are underway to interpret the linear genetic information of DNA in terms of function structure and control of biological process the systematic identification and quantification of expressed protein has proven particularly powerful in this regard large scale protein identification is usually achieved by automated liquid chromatography tandem mass spectrometry of complex peptide mixtures and sequence database searching of the resulting spectra aebersold and goodlett chem rev As generating large numbers of sequence specific mass spectra collision induced dissociation CID spectra has become routine operation research has shifted from the generation of sequence database search results to their validation here we describe in detail novel probabilistic model and score function that ranks the quality of the match between tandem mass spectral data and peptide sequence in database We document the performance of the algorithm on reference data set and in comparison with another sequence database search tool the software is publicly available for use and evaluation at http www systemsbiology org research software proteomics probID

this study applies new quantitative proteomics technology to the analysis of the function of the myc oncoprotein in mammalian cell employing isotope coded affinity tag ICAT reagent labeling and tandem mass spectrometry the global pattern of protein expression in rat myc null cell was compared with that of myc plus cell myc null cell in which myc has been introduced to generate differential protein expression catalog expression differences among many functionally related protein were identified including reduction of proteases induction of protein synthesis pathways and upregulation of anabolic enzymes in myc plus cell which are predicted to lead to increased cell mass cell growth In addition reduction in the levels of adhesion molecules actin network protein and rho pathway protein were observed in myc plus cell leading to reduced focal adhesions and actin stress fibers as well as altered morphology these effects are dependent on the highly conserved myc box II region our results reveal novel cytoskeletal function for myc and indicate the feasibility of quantitative whole proteome analysis in mammalian cell

comprehensive proteome analysis requires the identification and quantification of the protein in samples consisting of thousands of protein spanning range of abundance of several orders of magnitude the currency of proteome analysis by mass spectrometry is the peptide generated by protein proteolysis the high sample complexity of such samples requires large separation capacity which is commonly achieved by fractionation of the mixture followed by further serial separations of each fraction the sample throughput of proteome analysis is therefore limited by the need to sequentially process large numbers of samples We have developed novel four plexed microcapillary liquid chromatography system for automated high throughput separation of complex peptide samples the system supports the concurrent separation of four different samples by directing identically split solvent gradient flows into four microcapillary C18 columns the simple design of the system achieves multiplexed separation without the need for extra solvent pumps peak resolution reproducibility and parallel separating capacity of the system were investigated using standard peptide the applicability of the system to high throughput protein expression profiling was demonstrated in qualitative and quantitative analyses of protein expression in cerevisiae grown on two different carbon sources using the isotope coded affinity tag ICAT reagent and matrix assisted desorption ionization quadrupole time of flight mass spectrometry

We examined the utility of gas phase fractionation GPF in the dimension to increase proteome coverage and reproducibility of peptide ion selection by direct microliquid chromatography electrospray ionization tandem mass spectrometry µLC ESI MS MS analysis of the peptide produced by proteolytic digestion of unfractionated protein from yeast whole cell lysate and in peroxisomal membrane protein fraction derived from isolated yeast peroxisomes We also investigated GPF in the relative ion intensity dimension and propose denoting the two types of GPF as GPFm and GPFRI comparison of results of direct µLC ESI MS MS analysis of the unfractionated mixture of peptide from proteolysis of yeast whole cell lysate by DD ion selection from in triplicate and GPFm from and produced the following results more protein were identified by GPFm for an equal amount of effort µLC ESI MS MS and ii protein identified by GPFm had lower average codon bias value use of GPFRI identified more protein per unit scanned than GPFm or triplicate analysis over wide range after tryptic digestion of all the protein from discontinuous nycodenz gradient fraction known to be enriched with yeast peroxisomal membrane protein we detected of known peroxisomal protein using GPFm but only using standard wide range survey scan

using coimmunoprecipitation and tandem mass spectrometry we identify INSIG as an ER protein that binds the sterol sensing domain of SREBP cleavage activating protein SCAP and facilitates retention of the SCAP SREBP complex in the ER In sterol depleted cell SCAP escorts SREBPs from ER to golgi for proteolytic processing thereby allowing SREBPs to stimulate cholesterol synthesis sterols induce binding of SCAP to INSIG as determined by blue native PAGE and this is correlated with the inhibition of SCAP exit from the ER overexpression of INSIG increases the sensitivity of cell to sterol mediated inhibition of SREBP processing mutant SCAP Y298C fails to bind INSIG and is resistant to sterol mediated inhibition of ER exit By facilitating sterol dependent ER retention of SCAP INSIG plays central role in cholesterol homeostasis

the proteomics analysis reported here shows that major cellular response to oxidative stress is the modification of several peroxiredoxins An acidic form of the peroxiredoxins appeared to be systematically increased under oxidative stress conditions peroxiredoxins are enzymes catalyzing the destruction of peroxides In doing so reactive cysteine in the peroxiredoxin active site is weakly oxidized disulfide or sulfenic acid by the destroyed peroxides cellular thiols thioredoxin are used to regenerate the peroxiredoxins to their active state tandem mass spectrometry was carried out to characterize the modified form of the protein produced in vivo by oxidative stress the cysteine present in the active site was shown to be oxidized into cysteic acid leading to an inactivated form of peroxiredoxin this strongly suggested that peroxiredoxins behave as dam upon oxidative stress being both important peroxide destroying enzymes and peroxide targets results obtained in primary culture of leydig cell challenged with tumor necrosis factor suggested that this oxidized native balance of peroxiredoxin may play an active role in resistance or susceptibility to tumor necrosis factor induced apoptosis

metabolism of the common industrial gas tetrafluoroethylene in mammals results in the formation of tetrafluoroethyl cysteine TFEC which can be bioactivated by mitochondrial lyase commonly referred to as lyase the resultant reactive intermediate difluorothioacetyl fluoride DFTAF is potent thioalkylating and protein modifying species previously we have identified mitochondrial HSP70 HSP60 aspartate aminotransferase and the E2 and E3 subunits of the ketoglutarate dehydrogenase aKGDH complex as specific protein structurally modified during this process moreover functional alterations to the aKGDH complex were also detected and implicated in the progression of injury We report here the identification by tandem mass spectrometry and functional characterization of the final remaining major protein species modified by DFTAF previously designated as P99 unk as mitochondrial aconitase aconitase activity was maximally inhibited by in renal homogenates after exposure to TFEC In comparison to aKGDH aconitase inhibition up to in cell culture model for TFEC mediated cytotoxicity was greater and preceded aKGDH inhibition indicating that aconitase modification may constitute an early event in TFEC mediated mitochondrial damage and cell death these findings largely define the initial lesion of TFEC mediated cell death and also have implications for the modeling of mitochondrial enzymatic architecture and the localization and identity of renal mitochondrial cysteine conjugate lyase

the adaptation of sequence of chemical reactions to solid phase format has been essential to the automation reproducibility and efficiency of number of biotechnological process including peptide and oligonucleotide synthesis and sequencing here we describe method for the site specific stable isotopic labeling of cysteinyl peptide in complex peptide mixtures through solid phase capture and release process and the concomitant isolation of the labeled peptide the recovered peptide were analyzed by microcapillary liquid chromatography and tandem mass spectrometry µLC MS MS to determine their sequence and relative quantities the method was used to detect galactose induced changes in protein abundance in the yeast saccharomyces cerevisiae side by side comparison with the isotope coded affinity tag ICAT method demonstrated that the solid phase method for stable isotope tagging of peptide is comparatively simpler more efficient and more sensitive

covalent attachment of ubiquitin is well known to target protein for degradation here mass spectrometry was used to identify the site of ubiquitination in gpa1 the protein subunit in yeast saccharomyces cerevisiae the modified residue is located at lys165 within the helical domain of Ga region of unknown function substitution of lys165 with arg gpa1K165R results in substantial decrease in ubiquitination In addition yeast expressing the gpa1K165R mutant are moderately resistant to pheromone in growth inhibition assay phenotype consistent with enhanced Ga signaling activity these findings indicate that the helical domain may serve to regulate the turnover of gpa1

following cell antigen receptor BCR ligation the cytoplasmic domains of immunoglobulin iga and Igß recruit syk to initiate signaling cascades the coupling of syk to several distal substrates requires linker protein BLNK however the mechanism by which BLNK is recruited to the BCR is unknown using chimeric receptors with wild type and mutant iga cytoplasmic tails we show that the non immunoreceptor tyrosine based activation motif ITAM tyrosines Y176 and Y204 are required to activate BLNK dependent pathways subsequent analysis demonstrated that BLNK bound directly to phospho Y204 and that fusing BLNK to mutated iga reconstituted downstream signaling events moreover ligation of the endogenous BCR induced Y204 phosphorylation and BLNK recruitment these data demonstrate that the non ITAM tyrosines of iga couple syk activation to BLNK dependent pathways

using an integrated genomic and proteomic approach we have investigated the effects of carbon source perturbation on steady state gene expression in the yeast saccharomyces cerevisiae growing on either galactose or ethanol for many gene significant differences between the abundance ratio of the messenger RNA transcript and the corresponding protein product were observed insights into the perturbative effects on gene involved in respiration energy generation and protein synthesis were obtained that would not have been apparent from measurements made at either the messenger RNA or protein level alone illustrating the power of integrating different types of data obtained from the same sample for the comprehensive characterization of biological system and process

after infection of mice with the intracellular bacterium mycobacterium bovis bacillus calmette Guérin macrophages recovered from the peritoneal cavity display classical signs of immune activation We have identified member of the serine protease inhibitor serpin family which is highly induced in macrophages during bacillus calmette Guérin infection serpin 2a spi2a expression is also induced in macrophages in vivo during infection with salmonella typhimurium and listeria monocytogenes and in vitro by variety of bacteria and bacterial products the cytokine IFN also induces spi2a expression in macrophages and this induction is synergistic with bacterial products We also demonstrate here that ubiquitin homolog IFN stimulated gene of kDa ISG15 is strongly induced during in vitro and in vivo activation of macrophages and that it conjugates to spi2a in activated macrophages the ISG15 spi2a conjugates were identified by tandem mass spectrometry and contained spi2a conjugated to either one or two molecules of ISG15 whereas spi2a was induced by either bacterial products or IFN ISG15 was induced only by bacterial products although many protein targets have been described for ubiquitin conjugation spi2a is the first ISG15 modified protein to be reported macrophage activation is accompanied by the activation of variety of proteases It is of interest that member of the serine protease inhibitor family is concomitantly induced and modified by ubiquitin like protein

bad is pro apoptotic member of the bcl family of protein that is thought to exert death promoting effect by heterodimerization with bcl XL nullifying its anti apoptotic activity growth factors may promote cell survival at least partially through phosphorylation of bad at one or more of ser or our previous work showed that bad is also phosphorylated in response to cytokines at another site which we now identify as ser the functional role of this novel phosphorylation site was assessed by site directed mutagenesis and analysis of the pro apoptotic function of bad in transiently transfected HEK293 and COS cell or by stable expression in the cytokine dependent cell line MC In general mutation of ser to ala results in protein with increased ability to induce apoptosis similar to the S112A mutant mutation of ser to asp mimicking constitutively phosphorylated site results in protein that is virtually unable to induce apoptosis similarly the S112A S170D double mutant does not cause apoptosis in HEK293 and MC cell lines these data strongly suggest that phosphorylation of bad at ser is critical event in blocking the pro apoptotic activity of bad

the effectiveness of proteome wide protein identification and quantitative expression profiling is dependent on the ability of the analytical methodologies employed to routinely obtain information on low abundance protein as these are frequently of great biological importance two dimensional gel electrophoresis the traditional method for proteome analysis has proven to be biased toward highly expressed protein recently two dimensional chromatography of the complex peptide mixtures generated by the digestion of unseparated protein samples has been introduced for the identification of their components and isotope coded affinity tags ICAT have been introduced to allow for accurate quantification of the components of protein mixtures by mass spectrometry here we demonstrate that the combination of isotope coded affinity protein tags and multidimensional chromatography mass spectrometry of tryptic peptide mixtures is capable of detecting and quantifying protein of low abundance in complex samples

quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately reproducibly and comprehensively identify and quantify the protein contained in biological samples We describe new strategy for quantitative protein profiling that is based on the separation of protein labeled with isotope coded affinity tag reagents by two dimensional gel electrophoresis and their identification and quantification by mass spectrometry the method is based on the observation that protein labeled with isotopically different isotope coded affinity tag reagents precisely co migrate during two dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel By analyzing changes in the proteome of yeast saccharomyces cerevisiae induced by metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple protein migrate to the same gel coordinates the method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post translationally modified forms of protein and is therefore expected to find wide application in proteomics research

bovine aortic smooth muscle cell BASMC cultures undergo mineralization on addition of the organic phosphate donor glycerophosphate ßGP mineralization is characterized by apatite deposition on collagen fibrils and the presence of matrix vesicles as has been described in calcified vascular lesions in vivo as well as in bone and teeth In the present study we used this model to investigate the molecular mechanisms driving vascular calcification We found that BASMCs lost their lineage markers SM22a and smooth muscle actin within days of being placed under calcifying conditions conversely the cell gained an osteogenic phenotype as indicated by an increase in expression and DNA binding activity of the transcription factor core binding factor a1 cbfal moreover gene containing the cbfal binding site OSE2 including osteopontin osteocalcin and alkaline phosphatase were elevated the relevance of these in vitro findings to vascular calcification in vivo was further studied in matrix GLA protein null MGP mice whose arteries spontaneously calcify We found that arterial calcification was associated with similar loss in smooth muscle markers and gain of osteopontin and cbfal expression these data demonstrate novel association of vascular calcification with smooth muscle cell phenotypic transition in which several osteogenic protein including osteopontin osteocalcin and the bone determining factor cbfal are gained the findings suggest positive role for SMCs in promoting vascular calcification

plasma membranes of most cell types are thought to contain microdomains commonly referred to as lipid rafts biochemically distinct from bulk plasma membrane and apparently enriched for protein involved in signal transduction In cell it is believed that lipid rafts aggregate at the site of cell receptor engagement and act as foci for initiation of the signaling process In order to gain insight into the possible functioning of lipid rafts we applied microcapillary liquid chromatography electrospray ionization tandem mass spectrometry µLC ESI MS MS methodologies to the identification of protein which co purified with lipid rafts following isolation of lipid rafts as triton insoluble low density membrane fractions from jurkat cell tryptic digests were generated of electrophoretically resolved individual protein bands alternatively avidin affinity purification was used to isolate cysteine containing peptide from total tryptic digests of unseparated lipid raft protein following protein labeling with cysteine specific biotinylation reagent In both cases protein identifications were made by comparison of tandem mass spectra generated by µLC ESI MS MS with both protein and DNA sequence databases using sequest software protein identified essentially fell into two groups cytoskeletal protein and protein involved in signal transduction these findings are discussed in light of the current understanding of both lipid raft biology and signal transduction

present address geneva proteomics inc CP125 meyrin switzerland and institute for system biology rooesevelt way NE suite seattle WA CD28 delivers co stimulatory signal fortcell antigen receptor induced activation of cell through mechanism which remains mostly elusive to date In order to try and gain insight into CD28 function we therefore applied state of the art mass spectrometric protein identification technology to the analysis of CD28 immunoprecipitates prepared from jurkat cell We found that ethylmaleimide sensitive fusion protein NSF and other protein with sequence similarities to protein part of or implicated in vesicular protein sorting pathways were associated with CD28 in CD28 stimulationdependent manner furthermore ethylmaleimide treatment abolished the NSF CD28 interaction completely and blocked CD28 association with tyrosine phosphorylated kDa protein in the activated cell these results are suggestive of potential model for CD28 co stimulation regulated by an NSF catalyzed mechanism

proteome characterization using mass spectrometry is essential for the systematic investigation of biological system and for the study of gene function recent advances in this multifaceted field have occurred in four general areas protein and peptide separation methodologies selective labeling chemistries for quantitative measurement of peptide and protein abundances characterization of post translational protein modifications and instrumentation

the isotope coded affinity tag ICAT technology enables the concurrent identification and comparative quantitative analysis of protein present in biological samples such as cell and tissue extracts and biological fluids by mass spectrometry the initial implementation of this technology was based on microcapillary chromatography coupled on line with electrospray ionization tandem mass spectrometry this implementation lacked the ability to select protein for identification based on their relative abundance and therefore to focus on differentially expressed protein In order to improve the sample throughput of this technology we have developed two step approach that is focused on those protein for which the abundance changes between samples first new software program for the automated quantification of ICAT reagent labeled peptide analyzed by microcapillary electrospray ionization time of flight mass spectrometry determines those peptide that differ in their abundance and second these peptide are identified by tandem mass spectrometry using an electrospray quadrupole time of flight mass spectrometer and sequence database searching results from the application of this approach to the analysis of differentially expressed protein secreted from nontumorigenic human prostate epithelial cell and metastatic cancerous human prostate epithelial cell are shown copyright

the monoclonal antibody MAb A6H originally developed to fetal renal tissues was found to be highly reactive to renal cell carcinoma and was subsequently demonstrated to co stimulate subpopulation of cell the A6H antigen had not been identified heretofore antigen from detergent extracts of renal cell carcinoma cell was immunoabsorbed with A6H agarose and the resin bound protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE the antigen had molecular weight of approximately kDa as determined by western blots the kDa protein band was excised and subjected to in gel tryptic digestion and the resulting peptide were separated and analyzed by liquid chromatography tandem mass spectrometry LC MS MS the tandem mass spectra of the eluting peptide were used in combination with the SEQUEST computer program to search human national cancer institute NCI protein database for the identity of the protein the target antigen was shown to be dipeptidyl peptidase IV DPP IV which is also known as the cluster differentiation antigen CD26 flow analysis of the expression of the A6H antigen and of CD26 on cell and on peripheral blood lymphocytes supported the identification of the A6H antigen as DPP IV recognition that the A6H antigen is DPP IV CD26 afforded the opportunity to compare previous studies on A6H with those on other anti CD26 antibodies in terms of expression in cancer cell lines and various tissues and as co stimulators of cell activation

In most instances translation is regulated at the initiation phase when ribosome is recruited to the end of an mRNA the eIF4E binding protein 4E BPs interdict translation initiation by binding to the translation factor eIF4E and preventing recruitment of the translation machinery to mRNA the 4E BPs inhibit translation in reversible manner hypophosphorylated 4E BPs interact avidly with eIF4E whereas 4E BP hyperphosphorylation elicited by stimulation of cell with hormones cytokines or growth factors results in an abrogation of eIF4E binding activity We reported previously that phosphorylation of 4E BP1 on thr and thr is relatively insensitive to serum deprivation and rapamycin treatment and that phosphorylation of these residue is required for the subsequent phosphorylation of set of unidentified serum responsive sites here using mass spectrometry we identify the serum responsive rapamycin sensitive sites as ser and thr utilizing novel combination of two dimensional isoelectric focusing SDS PAGE and western blotting with phosphospecific antibodies we also establish the order of 4E BP1 phosphorylation in vivo phosphorylation of thr thr is followed by thr phosphorylation and ser is phosphorylated last finally we show that phosphorylation of ser and thr alone is insufficient to block binding to eIF4E indicating that combination of phosphorylation events is necessary to dissociate 4E BP1 from eIF4E

calcium calmodulin dependent protein kinase II CaM kinase II decoder of Ca2+ signals and cytosolic phospholipase A2 cPLA an enzyme involved in arachidonate release are involved in many physiological and pathophysiological process activation of CaM kinase II in norepinephrine stimulated vascular smooth muscle cell leads to activation of cPLA2 and arachidonic acid release surface plasmon resonance mass spectrometry and kinetic studies show that CaM kinase II binds to cPLA resulting in cPLA2 phosphorylation on ser and an increase in its enzymatic activity phosphopeptide mapping studies with cPLA2 from norepinephrine stimulated smooth muscle cell indicates that phosphorylation of cPLA2 on ser but not on ser or ser occurs in vivo this novel signaling pathway for arachidonate release is shown to be cPLA2 dependent by use of recently described and highly selective inhibitor of this enzyme

An approach to the systematic identification and quantification of the protein contained in the microsomal fraction of cell is described It consists of three steps preparation of microsomal fractions from cell or tissues representing different states covalent tagging of the protein with isotope coded affinity tag ICAT reagents followed by proteolysis of the combined labeled protein samples and isolation identification and quantification of the tagged peptide by multidimensional chromatography automated tandem mass spectrometry and computational analysis of the obtained data the method was used to identify and determine the ratios of abundance of each of protein contained in the microsomal fractions of naive and in vitrodifferentiated human myeloid leukemia HL cell the method and the new software tools to support it are well suited to the large scale quantitative analysis of membrane protein and other classes of protein that have been refractory to standard proteomics technology

RNA editing in kinetoplastid mitochondria occurs by series of enzymatic steps that is catalyzed by macromolecular complex four novel protein and their corresponding gene were identified by mass spectrometric analysis of purified editing complexes from trypanosoma brucei these four protein TbMP81 TbMP63 TbMP42 and TbMP18 contain conserved sequence to various degrees all four protein have sequence similarity in the terminus TbMP18 has considerable sequence similarity to the terminal region of TbMP42 and TbMP81 TbMP63 and TbMP42 contain zinc finger motif monoclonal antibodies that are specific for TbMP63 and TbMP42 immunoprecipitate in vitro RNA editing activities the protein are present in the immunoprecipitates and sediment at 20S along with the in vitro editing and RNA editing ligases TbMP52 and TbMP48 recombinant TbMP63 and TbMP52 coimmunoprecipitate these results indicate that these four protein are components of the RNA editing complex and that TbMP63 and TbMP52 can interact

the combination of isotope coded affinity tag ICAT reagents and tandem mass spectrometry constitutes new method for quantitative proteomics It involves the site specific covalent labeling of protein with isotopically normal or heavy ICAT reagents proteolysis of the combined labeled protein mixture followed by the isolation and mass spectrometric analysis of the labeled peptide the method critically depends on labeling protocols that are specific quantitative general robust and reproducible here we describe the systematic evaluation of important parameters of the labeling protocol and describe optimized labeling conditions the tested factors include the ICAT reagent concentration the influence of the protein SDS and urea concentrations on the labeling reaction and the reaction time We demonstrate that using the optimized conditions specific and quantitative labeling was achieved on standard protein as well as in complex protein mixtures such as yeast cell lysate

the antioxidant system of mitochondria are not well known using proteomics based approach we defined these mitochondrial antioxidant system and analyzed their response to oxidative stress It appears that the major mitochondrial antioxidant system is made of manganese superoxide dismutase on the one hand and of peroxiredoxin III mitochondrial thioredoxin and mitochondrial thioredoxin reductase on the other hand with the exception of thioredoxin reductase all these protein are induced by oxidative stress In addition change in the peroxiredoxin III pattern can also be observed

We have demonstrated the use of per methyl esterification of peptide for relative quantification of protein between two mixtures of protein and automated de novo sequence derivation on the same dataset protein mixtures for comparison were digested to peptide and resultant peptide methylated using either d0 or d3 methanol methyl esterification of peptide converted carboxylic acids such as are present on the side chains of aspartic and glutamic acid as well as the carboxyl terminus to their corresponding methyl esters the separate d0 and d3 methylated peptide mixtures were combined and the mixture subjected to microcapillary high performance liquid chromatography tandem mass spectrometry HPLC MS MS parent protein of methylated peptide were identified by correlative database searching of peptide tandem mass spectra ratios of protein in the two original mixtures could be calculated by normalization of the area under the curve for identical charge states of d0 to d3 methylated peptide An algorithm was developed that derived without intervention peptide sequence de novo by comparison of tandem mass spectra of d0 and d3 peptide methyl esters copyright

plasma membranes of most cell types are thought to contain microdomains commonly referred to as lipid rafts biochemically distinct from bulk plasma membrane apparently enriched for protein involved in signal transduction In cell it is believed that lipid rafts aggregate at the site of cell receptor engagement and act as foci for initiation of the signaling process In order to gain insight into the possible functioning of lipid rafts we applied microcapillary liquid chromatography electrospray ionization tandem mass spectrometry µLC ESI MS MS methodologies to the identification of protein which copurified with lipid rafts following isolation of lipid rafts as triton insoluble low density membrane fractions from jurkat cell tryptic digests were generated of individual protein bands resolved electrophoretically alternatively cysteine containing peptide were isolated from total tryptic digests of unseparated lipid raft protein following labeling with cysteine specific biotinylation reagent and avidin affinity purification In both cases protein identifications were made by comparison of tandem MS spectra generated by µLC ESI MS MS to both protein and DNA sequence databases using sequest software protein identified essentially fell into two groups cytoskeletal protein and protein involved in signal transduction these findings are discussed in the light of the current understanding of both lipid raft biology and signal transduction

A soluble phosphatidic acid preferring phospholipase A1 expressed in mature bovine testes but not in newborn calf testes may contribute to the formation or function of sperm here we incubated recombinant preparation of the phospholipase in vitro with several enzymes including protein kinase CK2 CK2 extracellular signal regulated kinase ERK2 and protein phosphatase 2A PP2A to identify effects that might be of regulatory importance in vivo major findings were that CK2 phosphorylated the phospholipase on serines and ERK2 phosphorylated the enzyme on serine there was cross antagonism between the reactions that phosphorylated serines and PP2A selectively hydrolyzed phosphate groups that were esterified to serines and CK2a formed stable MgATP MgGTP dependent complex with the phospholipase by novel mechanism and the complex showed reduced phospholipase activity and resembled complex identified in homogenates of macaque testis these results provide the first available information about the effects of reactions of phosphorylation and dephosphorylation on the behavior of the phospholipase shed light on properties of CK2a that may be required for the formation of complexes with its substrates and raise the possibility that complex containing CK2a and the phospholipase may play special biological role in the testis

We have identified and characterized an alternative RFC complex RFC ctf18p ctf8p dcc1p that is required for sister chromatid cohesion and faithful chromosome transmission ctf18p ctf8p and dcc1p interact physically in complex with rfc2p rfc3p rfc4p and rfc5p but not with rfc1p or rad24p deletion of CTF18 CTF8 or DCC1 singly or in combination ctf18 ctf8 dcc1 leads to sensitivity to microtubule depolymerizing drugs and severe sister chromatid cohesion defect furthermore temperature sensitive mutations in RFC4 result in precocious sister chromatid separation our results highlight novel function of the RFC protein and support model in which sister chromatid cohesion is established at the replication fork via polymerase switching mechanism and replication coupled remodeling of chromatin

We demonstrate an integrated approach to build test and refine model of cellular pathway in which perturbations to critical pathway components are analyzed using DNA microarrays quantitative proteomics and databases of known physical interactions using this approach we identify messenger RNAs responding to systematic perturbations of the yeast galactose utilization pathway provide evidence that approximately of detected protein are regulated posttranscriptionally and identify explicit physical interactions governing the cellular response to each perturbation We refine the model through further iterations of perturbation and global measurements suggesting hypotheses about the regulation of galactose utilization and physical interactions between this and variety of other metabolic pathways

reversible protein phosphorylation has been known for some time to control wide range of biological functions and activities thus determination of the site of protein phosphorylation has been an essential step in the analysis of the control of many biological system however direct determination of individual phosphorylation sites occurring on phosphoproteins in vivo has been difficult to date typically requiring the purification to homogeneity of the phosphoprotein of interest before analysis thus there has been substantial need for more rapid and general method for the analysis of protein phosphorylation in complex protein mixtures here we describe such an approach to protein phosphorylation analysis It consists of three steps selective phosphopeptide isolation from peptide mixture via sequence of chemical reactions phosphopeptide analysis by automated liquid chromatography tandem mass spectrometry LC MS MS and identification of the phosphoprotein and the phosphorylated residue by correlation of tandem mass spectrometric data with sequence databases By utilizing various phosphoprotein standards and whole yeast cell lysate we demonstrate that the method is equally applicable to serine threonine and tyrosine phosphorylated protein and is capable of selectively isolating and identifying phosphopeptides present in highly complex peptide mixture

the melanoma growth stimulatory activity growth regulated protein CXCL1 is constitutively expressed at high levels during inflammation and progression of melanocytes into malignant melanoma It has been shown previously that CXCL1 overexpression in melanoma cell is due to increased transcription as well as stability of the CXCL1 message the transcription of CXCL1 is regulated through several cis acting elements including Sp1 NF HMGI and the immediate upstream region IUR element nucleotides to which lies immediately upstream to the nuclear factor NF element previously it has been shown that the IUR is necessary for basal and cytokine induced transcription of the CXCL1 gene UV cross linking and southwestern blot analyses indicate that the IUR oligonucleotide probe selectively binds kDa protein In this study the IUR element has been further characterized We show here that proximity of the IUR element to the adjacent NF element is critical to its function as positive regulatory element using binding site oligonucleotide affinity chromatography we have selectively purified the kDa IUR mass spectrometry mass spectrometry matrix assisted laser desorption ionization time of flight spectroscopy and amino-acid analysis as well as microcapillary reverse phase chromatography electrospray ionization tandem mass spectrometry identified this protein as the kDa poly ADP ribose polymerase PARP1 furthermore aminobenzamide an inhibitor of PARP specific ADP ribosylation inhibits CXCL1 promoter activity and reduces levels of CXCL1 mRNA the data point to the possibility that PARP may be coactivator of CXCL1 transcription

the yeast mediator complex is required for transcription by RNA polymerase II pol II in vivo and in vitro this complex of over polypeptides associates with pol II and is recruited to transcription complexes at promoters previous isolations of yeast mediator containing complexes in different laboratories have identified several distinct complexes To identify the major forms of mediator in yeast mediator was isolated from nuclear extracts using two step chromatographic procedure avoiding ion exchange chromatography and high salt conditions to prevent dissociation of subunits during purification components of the mediator complexes were identified by mass spectrometry and western analysis the major form of mediator termed pol II med contained pol II and mediator including the srb8 module second lower molecular size complex was also identified termed mediator core medc which lacked pol II srbS rox3 nut1 and the rgr1 module both of these complexes were active in transcription in vitro although the medc complex had significantly lower activity and could compete with the activity of the pol II med complex in vitro

We describe an approach to the quantitative analysis of complex protein mixtures using MALDI quadrupole time of flight MALDI QqTOF mass spectrometer and isotope coded affinity tag reagents gygi et al nat biotechnol protein in mixtures are first labeled on cysteinyl residue using an isotope coded affinity tag reagent the protein are enzymatically digested and the labeled peptide are purified using multidimensional separation procedure with the last step being the elution of the labeled peptide from microcapillary reversed phase liquid chromatography column directly onto MALDI sample target after addition of matrix the sample spots are analyzed using MALDI QqTOF mass spectrometer by first obtaining mass spectrum of the peptide in each sample spot in order to quantify the ratio of abundance of pairs of isotopically tagged peptide followed by tandem mass spectrometric analysis to ascertain the sequence of selected peptide for protein identification the effectiveness of this approach is demonstrated in the quantification and identification of peptide from control mixture of protein of known relative concentrations and also in the comparative analysis of protein expression in saccharomyces cerevisiae grown on two different carbon sources

current method that are the basis for proteome analysis are reviewed focus is on mass spectrometric strategies for protein identification from biological matrixes computational approaches for searching sequence databases determination of protein expression levels and characterization of phosphoproteins and phosphopeptides

RNA editing in kinetoplastid mitochondria inserts and deletes uridylates at multiple sites in pre mRNAs as directed by guide RNAs this occurs by series of steps that are catalyzed by endoribonuclease terminal uridylyl transferase exouridylylase and RNA ligase activities multiprotein complex that contains these activities and catalyzes deletion editing in vitro was enriched from trypanosoma brucei mitochondria by sequential ion exchange and gel filtration chromatography followed by glycerol gradient sedimentation the complex size is approximately kDa and the purified fraction contains major polypeptides monoclonal antibody that was generated against the enriched complex reacts with an kDa protein and specifically immunoprecipitates in vitro deletion RNA editing activity the protein recognized by the antibody was identified by mass spectrometry and the corresponding gene designated TbMP52 was cloned recombinant TbMP52 reacts with the monoclonal antibody another novel protein TbMP48 which is similar to TbMP52 and its gene were also identified in the enriched complex these results suggest that TbMP52 and TbMP48 are components of the RNA editing complex

with the completion of rapidly increasing number of complete genomic sequence much attention is currently focused on how the information contained in sequence databases might be interpreted in terms of the structure function and control of biological system quantitative proteome analysis the global analysis of protein expression has been proposed as method to study steady state gene expression and perturbation induced changes here we discuss the rationale for quantitative proteome analysis highlight the limitations in the current standard technology and introduce new experimental approach to quantitative proteome analysis

proteomics can be defined as the analysis of biological process and system by the systematic analysis of large number of expressed protein for specific properties such as their identity quantity activity and molecular interactions over the past few years exceptional progress has been made towards developing mature technology for the identification and cataloging of the protein expressed in cell or tissue descriptive proteomics more recently efforts have focused on quantitative proteomics technology that can also capture the dynamics of biological system

phosphorylation of the nonstructural NS5A protein is highly conserved among hepatitis virus HCV genotypes however the precise site or sites of phosphorylation of NS5A have not been determined and the functional significance of phosphorylation remains unknown here we showed by two dimensional phosphopeptide mapping that protein kinase or kinases present in yeast insect and mammalian cell phosphorylated highly purified HCV genotype 1b NS5A from insect cell on identical serine residue We identified major phosphopeptide corresponding to amino-acid of the HCV 1b polyprotein by using negative ion electrospray ionization microcapillary high performance liquid chromatographymass spectrometry the elution time of the phosphopeptide determined by negative ion electrospray ionization mass spectrometry corresponded with the elution time of the majority of 32P label that was incorporated into the phosphopeptide by an in vitro kinase reaction subsequent analysis of the peak fraction by automated positive ion electrospray ionizationtandem mass spectrometry revealed that ser2194 was the major phosphorylated residue on the phosphopeptide Gp SPPSLASSSASQLSAPSLK substitution for ser2194 with ala resulted in the concomitant disappearance of major in vivo phosphorylated peptide ser2194 and surrounding amino-acid are highly conserved in all HCV genotypes suggesting NS5A phosphorylation at ser2194 may be an important mechanism for modulating NS5A biological functions

A p38 MAPK homolog mipk meiosis inhibited protein kinase was cloned from seastar oocytes this kDa protein shares approximately amino-acid identity with mammalian p38 isoforms mipk was one of the major tyrosine phosphorylated protein in immature oocytes arrested at the G2 transition of meiosis the tyrosine phosphorylation of mipk was increased in response to anisomycin heat and osmotic shock of oocytes during methyladenine induced oocyte maturation mipk underwent tyrosine dephosphorylation and remained dephosphorylated in mature oocytes and during the early mitotic cell divisions until approximately after fertilization At the time of differentiation and acquisition of phases in the developing embryos mipk was rephosphorylated on tyrosine In oocytes that were microinjected with mipk antisense oligonucleotides and subsequently were allowed to mature and become fertilized differentiation was blocked because mipK antisense oligonucleotides and dominant negative K62R mipk when microinjected into immature oocytes failed to induce germinal vesicle breakdown inhibition of mipk function was not sufficient by itself to cause oocyte maturation these findings point to putative role for mipk in cell cycle control as phase promoting factor

glycoproteins carrying linked acetylglucosamine glcNAc modifications have been isolated from wide range of organisms ranging from trypanosomes to humans interest in this modification is increasing as evidence accumulates that it is an abundant and transient modification that is dynamic and responsive to cellular stimuli concurrent advances in biological mass spectrometry MS have facilitated high sensitivity protein identification by tandem MS In this study we show that the lability of the glcNAc moiety to low energy collision in tandem MS offers means of distinguishing such peptide from others that are not modified the differential between the energy required to remove the glcNAc group and the energy required to fragment the peptide chain allows the glcNAc group to be detected and the peptide sequence and therefore the protein to be identified this technique thus allows the simultaneous detection and identification of glcNAc modified peptide even when present at low levels in complex mixtures the method was initially developed and validated using synthetic glcNAc modified peptide and then applied to the detection of an extremely low abundance glcNAc modified peptide from bovine crystallin We believe that with further development this assay system may prove to be useful tool for the direct investigation of intracellular glcNAc levels thus providing valuable insights into the physiological role of glcNAc modified protein

proteomics is the systematic analysis of the protein expressed by cell or tissue and mass spectrometry is its essential analytical tool In the past two years incremental advances in standard proteome technology have increased the speed of protein identification with higher levels of automation and sensitivity furthermore new approaches have provided landmark advances in determining functionally relevant properties of protein including their quantity and involvement within protein complexes

In the present study we identified positive transcriptional element within the rat Ha ras promoter previously known as Ha ras response element HRE and identified trans acting factor that binds HRE sequence in rat mammary cell To identify the binding protein we employed sequence specific DNA affinity chromatography amino-acid sequence analysis of the affinity purified protein was performed by tandem mass spectroscopy the results unexpectedly demonstrated that in rat mammary cell CArG box binding factor CBF is the major protein species that bind specifically to the rat and human HRE sequence with high affinity the affinity of CBF binding to HRE was significantly higher than to the CArG box described as recognition sequence for CBF protein transient transfection assay using reporter plasmids verified that mutations within the HRE that disrupt binding of CBF also reduced the activity of the rat Ha ras promoter despite the fact that the HRE within the Ha ras promoter resembles binding site for ets transcription factors we did not detect the binding of ets related protein to the rat HRE in BICR M1Rk cell We further demonstrated correlation between the presence of HRE binding activity and induction of Ha ras mRNA expression following serum stimulation in the mammary carcinoma cell line taken together our results suggest that CBF may play an important role in transcriptional regulation of Ha ras promoter activity during normal mammary cell growth and carcinogenesis

An electrophoretic method has been developed for the extraction of peptide following in gel digests of SDS PAGE separated protein during electroextraction the peptide are trapped on strong cation exchange microcartridge before analysis by capillary LC ESI tandem mass spectrometry the spectra obtained by tandem mass spectrometry are searched directly against protein database for identification of the protein from which the peptide originated By minimizing surface exposure of the peptide during electroextraction reduction of the detection limits for protein identification is realized the performance of the peptide electroextraction was compared directly with the standard extraction method for in gel protein digests using standard dilution series of phosphorylase and carbonic anhydrase separated by SDS PAGE the lowest gel loading in which phosphorylase was identified using the standard extraction method was ng or fmol and the lowest gel loading in which phosphorylase was identified using electroextraction was ng or fmol the design of the microextxaction cartridge allows for direct interfacing with capillary LC which is crucial for maintaining low detection limits furthermore this method can be used for high throughput proteomics since it can be easily multiplexed and requires only voltage control and low pressures psi for operation We believe that peptide electroextraction is significant advance for identification of protein separated by one dimensional or two dimensional gel electrophoresis as it can be easily automated and requires less protein than conventional method

proteome analysis is most commonly accomplished by combination of two dimensional gel electrophoresis 2DE to separate and visualize protein and mass spectrometry MS for protein identification although this technique is powerful mature and sensitive questions remain concerning its ability to characterize all of the elements of proteome In the current study more than features were visualized by silver staining narrow pH range 2D gel in which mg of total soluble yeast protein was separated fifty spots migrating to region of cm2 were subjected to MS protein identification despite the high sample load and extended electrophoretic separation protein from gene with codon bias values of lt lower abundance protein were not found even though fully one half of all yeast gene fall into that range protein from gene with codon bias values of lt were found however if protein amounts exceeding the capacity of 2DE were fractionated and analyzed We conclude that the large range of protein expression levels limits the ability of the 2DE MS approach to analyze protein of medium to low abundance and thus the potential of this technique for proteome analysis is likewise limited

proteome analysis is most commonly accomplished by the combination of two dimensional gel electrophoresis for protein separation visualization and quantification and mass spectrometry for protein identification over the past year exceptional progress has been made towards developing new technology base for the precise quantification and identification of protein in complex mixtures that is quantitative proteomics

the effect of adjuvant arthritis AA on the pattern of rat serum protein includes the upregulation of haptoglobin orosomucoid a2 macroglobulin serine protease inhibitor thiostatin a1 antitrypsin reactive protein and the downregulation of kallikrein binding protein a1 inhibitor III apolipoprotein a2 HS glycoprotein albumin apolipoprotein IV transthyretin and transferrin minor changes are observed for Gc globulin ceruloplasmin and a1 macroglobulin AA thus grossly resembles the acute inflammatory response elicited by the injection of turpentine although the changes in the levels of negative acute phase protein APP are smaller in acute inflammation indomethacine and ibuprofen inhibit the effects of arthritis on the synthesis of rat serum protein in different ways the former is on average three times as effective as the latter each drug interferes differently with different protein In animals without AA both nonsteroidal anti inflammatory drugs NSAID mimick the inflammatory pattern to certain extent with more effect on the negative than on the positive APPs overall the shifts in serum protein levels parallel changes in inflammatory parameters such as joint swelling and serum interleukin IL activity protein quantitation after two dimensional electrophoresis DE reveals some effects of the drugs per se which escape detection by other routine tests

It has become apparent that many intracellular signaling process involve the dynamic reorganization of cellular protein into complex signaling assemblies that have specific subunit composition function and subcellular location since the elements of such assemblies interact physically multiprotein signaling complexes can be isolated and analyzed recent technical advances in highly sensitive protein identification by electrospray tandem mass spectrometry have dramatically increased the sensitivity with which such analyses can be performed the cell antigen receptor TCR is an oligomeric transmembrane protein complex that is essential to cell recognition and function the extracellular protein domains are responsible for ligand binding while intracellular domains generate and transduce signals in response to specific receptor ligand interactions We used microbore capillary chromatography tandem mass spectrometry to investigate the composition of the TCR protein complex isolated from resting and activated cell of the murine cell line CD11 We identified all the previously known subunits of the TCR CD3 complex as well as protein previously not known to associate with the TCR the catalytic activities of some of these protein could potentially be used to interfere pharmacologically with TCR signaling

several method have been developed for the comprehensive analysis of gene expression in complex biological system generally these procedures assess either portion of the cellular transcriptome or portion of the cellular proteome each approach has distinct conceptual and methodological advantages and disadvantages We have investigated the application of both method to characterize the gene expression pathway mediated by androgens and the androgen receptor in prostate cancer cell this pathway is of critical importance for the development and progression of prostate cancer Of clinical importance modulation of androgens remains the mainstay of treatment for patients with advanced disease To facilitate global gene expression studies we have first sought to define the prostate transcriptome by assembling and annotating prostate derived expressed sequence tags ESTs total of prostate ESTs were assembled into set of clusters putatively representing distinct transcripts these clusters were used to construct cDNA microarrays suitable for examining the androgen response pathway at the level of transcription the expression of gene was found to be induced by androgens this cohort included known androgen regulated gene such as prostate specific antigen PSA and several novel complementary DNAs cDNAs protein expression profiles of androgen stimulated prostate cancer cell were generated by two dimensional electrophoresis DE mass spectrometric analysis of androgen regulated protein in these cell identified the metastasis suppressor gene NDKA nm23 finding that may explain marked reduction in metastatic potential when these cell express functional androgen receptor pathway

collagen XI is heterotrimeric molecule found predominantly in heterotypic cartilage fibrils where it is involved in the regulation of fibrillogenesis this function is thought to involve the complex terminal domain the goal of this current study was to examine its structural organization to further elucidate the regulatory mechanism the amino propeptide a1 npp alone or with isoforms of the variable region were recombinantly expressed and purified by affinity and molecular sieve chromatography cys cys and cys cys disulfide bonds were detected by liquid chromatography tandem mass spectrometry this pattern is identical to the homologous a2 npp indicating that the recombinant protein were folded correctly anomalous elution on molecular sieve chromatography suggested that the variable region was extended which was confirmed using rotary shadowing the a1 npp formed globular head and the variable region an extended tail circular dichroism spectra analysis determined that the a1 npp comprised sheet whereas the variable region largely comprised non periodic structure taken together these results imply that the a1 npp cannot be accommodated within the core of the fibril and that the variable region and or minor helix facilitates its exclusion to the fibril surface this provides further support for regulation of fibril diameter by steric hindrance or by interactions with other matrix components that affect fibrillogenesis

A method for rapid and unambiguous identification of protein by sequence database searching using the accurate mass of single peptide and specific sequence constraints is described peptide masses were measured using electrospray ionization fourier transform ion cyclotron resonance mass spectrometry to an accuracy of ppm the presence of cysteine residue within peptide sequence was used as database searching constraint to reduce the number of potential database hits cysteine containing peptide were detected within mixture of peptide by incorporating chlorine into general alkylating reagent specific for cysteine residue secondary search constraints included the specificity of the protease used for protein digestion and the molecular mass of the protein estimated by gel electrophoresis the natural isotopic distribution of chlorine encoded the cysteine containing peptide with distinctive isotopic pattern that allowed automatic screening of mass spectra the method is demonstrated for peptide standard and unknown protein from yeast lysate using all possible yeast open reading frames as database As judged by calculation of codon bias low abundance protein were identified from the yeast lysate using this new method but not by traditional method such as tandem mass spectrometry via data dependent acquisition or mass mapping

A simple calculation using the radioactive decay of 32P incorporated into protein during in vitro kinase reactions is described that allows the overall stoichiometry of phosphorylation for the substrate protein or peptide to be calculated prior to using techniques such as diagnostic ion scanning to identify the molecular weight of an unknown phosphopeptide in complex mixture followed by tandem mass spectrometry MS MS to locate the phosphorylated residue within the phosphopeptide such calculations are predictive of the chances for successful characterization by these method An example of estimating the stoichiometry of peptide phosphorylation will be presented along with calculations that predict when adequate phosphopeptide is present in any given spot on the thin layer chromatography TLC plates used for two dimensional phosphopeptide 2DPP mapping to allow extraction and complete characterization by MS MS john wiley and sons ltd

the eukaryotic translation initiation factor 4G eIF4G protein play critical role in the recruitment of the translational machinery to mRNA the eIF4Gs are phosphoproteins however the location of the phosphorylation sites how phosphorylation of these protein is modulated and the identity of the intracellular signaling pathways regulating eIF4G phosphorylation have not been established In this report two dimensional phosphopeptide mapping demonstrates that the phosphorylation state of specific eIF4GI residue is altered by serum and mitogens phosphopeptides resolved by this method were mapped to the terminal one third of the protein mass spectrometry and mutational analyses identified the serum stimulated phosphorylation sites in this region as serines and phosphoinositide kinase PI3K inhibitors and rapamycin an inhibitor of the kinase FRAP mTOR FKBP12 rapamycin associated protein mammalian target of rapamycin prevent the serum induced phosphorylation of these residue finally the phosphorylation state of terminally truncated eIF4GI protein acquires resistance to kinase inhibitor treatment these data suggest that the kinases phosphorylating serines and are not directly downstream of PI3K and FRAP mTOR but that the accessibility of the terminus to kinases is modulated by this pathway

reactive oxygen species have been implicated in the pathogenesis of atherosclerosis and hypertension in part by promoting vascular smooth muscle cell VSMC growth We have previously shown that LY83583 generator of activated extracellular signal regulated kinases ERK1 with early min and late peaks and stimulated VSMC growth To investigate whether secreted oxidative stress induced factors termed SOXF from VSMC were responsible for late ERK1 activation in response to LY83583 we purified putative SOXF protein from conditioned medium of LY83583 exposure by sequential chromatography based on activation of ERK1 protein identified by capillary chromatography electrospray ionization tandem mass spectrometry and data base searching included heat shock protein HSP90 and cyclophilin western blot analysis of conditioned medium showed specific secretion of HSP90 but not HSP90 immunodepletion of HSP90 from conditioned medium significantly inhibited conditioned medium induced ERK1 activation human recombinant HSP90 reproduced the effect of conditioned medium on ERK1 activation these results show that brief oxidative stress causes sustained release of protein factors from VSMC that can stimulate ERK1 these factors may be important mediators for the effects of reactive oxygen species on vascular function

regulators of protein signaling RGS protein are well known to accelerate protein GTpase activity in vitro and to promote protein desensitization in vivo less is known about how RGS protein are themselves regulated To address this question we purified the RGS in yeast sst2 and used electrospray ionization mass spectrometry to identify post translational modifications this analysis revealed that sst2 is phosphorylated at ser and that phosphorylation occurs in response to pheromone stimulation ser lies within consensus mitogen activated protein MAP kinase phosphorylation site pro ser pro phosphorylation is blocked by mutations in the MAP kinase gene FUS3 KSS1 as well as by mutations in components needed for MAP kinase activation STE11 STE7 STE4 STE18 phosphorylation is also blocked by replacing ser with ala asp or glu but not thr these point mutations do not alter pheromone sensitivity as determined by growth arrest and reporter transcription assay however phosphorylation appears to slow the rate of sst2 degradation these findings indicate that the protein regulated MAP kinase in yeast can act as feedback regulator of sst2 itself regulator of protein signaling

parasite encoded membrane protein translocated to the surface of infected erythrocytes or in specialized vesicles underneath maurer clefts play key role in the sexual life cycle of plasmodium falciparum malaria causing protozoan by mediating key steps such as red blood cell invasion sequestration of infected cell in microcapillaries and red blood cell rupture large scale analysis of these membrane protein would therefore be of great help to gain knowledge of the different stages of the plasmodium falciparum life cycle In order to be able to detect and identify parasite encoded protein directed to the red blood cell membrane we first defined the conditions required for optimal extraction and separation of normal red blood cell ghost protein by two dimensional gel electrophoresis these conditions included the use of urea thiourea and new zwitterionic detergentS in the extraction and isoelectric focusing media the optimized conditions were then applied to analyze normal and falciparum infected red blood cell ghosts several protein spots were found only in infected ghosts and are expected to represent parasite encoded protein these protein are currently under investigation

We describe site http users unimi it ratserum homeframed html with clickable maps of serum protein of control and inflamed rats as well as quantitative data on the expression of such serum protein under varying physiological and experimental conditions this information enhances the value of minimally invasive techniques thus reducing the number of animals to be treated and eventually sacrificed in pharmacological toxicological research projects

two ribosomat protein S6 kinases pp52 S6K and pp70 S6K of the p70 S6 kinase family were markedly activated during meiotic maturation of pisaster ochraceus sea star oocytes rapid protocol was developed for the purification from the oocyte cytosol of pp52 S6K by fold with specific enzyme activity of µmol per min per mg the purified enzyme apparently featured the and terminal regions of pp70 S6K as it immunoreacted with antibodies directed to peptide patterned after these amino-acid sequence in mammalian pp70 S6K pp52 S6K was inhibited by fluoride IC50 mM but was relatively insensitive to glycerolphosphate EGTA dithiothreitol spermine heparin NaCl and metal ions such as Mn2+ Zn2+ and Ca2+ the consensus sequence for substrate phosphorylation was determined to be RXXSXR which was partially distinct from mammalian p70 S6K in its requirement for an amino terminal arginine phosphorylation of ribosomal protein S6 by p52 S6K occurred exclusively on serine on at least five tryptic peptide inhibition of sea star p52 S6K phosphotransferase activity after treatment with protein serine threonine phosphatases confirmed that p52 S6K was still regulated by phosphorylation the sea star S6 kinase was purified to near homogeneity with the regulatory and catalytic subunits of protein serine phosphatase 2A and the heat shock protein the association of an S6 kinase with phosphatase 2A was confirmed by coimmunoprecipitation of S6 kinase activity with phosphatase 2A specific antibodies the purified S6 kinase and the sea star oocyte system will be useful for analysis of upstream and downstream signaling events that lead to phosphorylation of the S6 protein and other targets

endothelial cell release nitric oxide NO acutely in response to increased laminar fluid shear stress and the increase is correlated with enhanced phosphorylation of endothelial nitric oxide synthase eNOS phosphoamino acid analysis of eNOS from bovine aortic endothelial cell labeled with 32P orthophosphate demonstrated that only phosphoserine was present in eNOS under both static and flow conditions fluid shear stress induced phosphate incorporation into two specific eNOS tryptic peptide as early as after initiation of flow the flow induced tryptic phosphopeptides were enriched separated by capillary electrophoresis with intermittent voltage drops also known as peak parking and analyzed by collision induced dissociation in tandem mass spectrometer two phosphopeptide sequence determined by tandem mass spectrometry TQpSFSLQER and KLQTRPpSPGPPPAEQLLSQAR were confirmed as the two flow dependent phosphopeptides by co migration with synthetic phosphopeptides because the sequence RIR TQpSFSLQER contains consensus substrate site for protein kinase PKB or akt we demonstrated that LY294002 an inhibitor of the upstream activator of PKB phosphatidylinositol kinase inhibited flow induced eNOS phosphorylation by and NO production by finally PKB phosphorylated eNOS in vitro at the same site phosphorylated in the cell and increased eNOS enzymatic activity by fold

delivery of protein and peptide to electrospray ionization mass spectrometers ESI MS has been demonstrated using glass and quartz microfabricated devices this paper reports the construction and use of poly dimethylsiloxane PDMS microfabricated soft polymer devices with mass spectrometry for protein analysis the PDMS devices were fabricated using replica molding against patterned photoresist generated by photolithographic techniques the PDMS devices were connected to the mass spectrometer via derivatized transfer capillary and samples were transferred by electroosmotic pumping the formulation of PDMS was optimized for compatibility with ESI and the devices were tested for performance the practical application of PDMS devices was demonstrated by the identification of rat serum albumin separated by gel electrophoresis extended contact of the sample with the surface of the PDMS device did not significantly affect the sample analysis and the limit of detection for samples run on PDMS device was comparable to the limit of detection achieved on glass devices this study suggests that PDMS devices fabricated using replica molding are compatible with ESI MS this will potentially lead to the construction of inexpensive microfabricated devices with complex designs and advanced functionalities

We describe an approach for the accurate quantification and concurrent sequence identification of the individual protein within complex mixtures the method is based on class of new chemical reagents termed isotope coded affinity tags ICATs and tandem mass spectrometry using this strategy we compared protein expression in the yeast saccharomyces cerevisiae using either ethanol or galactose as carbon source the measured differences in protein expression correlated with known yeast metabolic function under glucose repressed conditions the method is redundant if multiple cysteinyl residue are present and the relative quantification is highly accurate because it is based on stable isotope dilution techniques the ICAT approach should provide widely applicable means to compare quantitatively global protein expression in cell and tissues

We have developed rapid and efficient way of stretching DNA and denatured protein molecules for detection by fluorescence microscopy and atomic force microscopy AFM In the described method viscous drag created by transient rotational flow stretches randomly coiled DNA molecules or denatured protein stretching is achieved by dispensing droplet of sample solution containing DNA or denatured protein on MgCl2 soaked mica surface We present fluorescent images of straightened DNA molecules and AFM images of stress sheared reduced von willebrand factor as well as straightened DNA the described quick and reliable spin stretching technique will find wide applications in the analysis of single biopolymer molecules

the Na+ H+ exchanger isoform NHE is the key member of family of exchangers that regulates intracellular pH and cell volume activation of NHE by growth factors is rapid correlates with increased NHE phosphorylation and cell alkalinization and plays role in cell cycle progression By two dimensional tryptic peptide mapping of immunoprecipitated NHE we identify serine as the major serum stimulated amino-acid mutation of serine to alanine had no effect on acid stimulated Na+ H+ exchange but completely prevented the growth factor mediated increase in NHE affinity for H+ In addition we show that p90 ribosomal S6 kinase p90 RSK is key NHE kinase since p90 RSK phosphorylates NHE serine stoichiometrically in vitro and transfection with kinase inactive p90 RSK inhibits serum induced phosphorylation of NHE serine in transfected cell these findings establish p90 RSK as serum stimulated NHE kinase and mediator of increased Na+ H+ exchange in vivo

the multisubunit eukaryotic translation initiation factor eIF 4F recruits 40S ribosomal subunits to the end of mRNA the eIF4F subunit eIF4E interacts directly with the mRNA cap structure assembly of the eIF4F complex is inhibited by family of repressor polypeptides the eIF4E binding protein 4E BPs binding of the 4E BPs to eIF4E is regulated by phosphorylation hypophosphorylated 4E BP isoforms interact strongly with eIF4E whereas hyperphosphorylated isoforms do not 4E BP1 is hypophosphorylated in quiescent cell but is hyperphosphorylated on multiple sites following exposure to variety of extracellular stimuli the PI3 kinase akt pathway and the kinase FRAP mTOR signal to 4E BP1 FRAP mTOR has been reported to phosphorylate 4E BP1 directly in vitro however it is not known if FRAP mTOR is responsible for the phosphorylation of all 4E BP1 sites nor which sites must be phosphorylated to release 4E BP1 from eIF4E To address these questions recombinant FRAP mTOR protein and FRAP mTOR immunoprecipitate were utilized in in vitro kinase assay to phosphorylate 4E BP1 phosphopeptide mapping of the ill vitro labeled protein yielded two 4E BP1 phosphopeptides that comigrated with phosphopeptides produced in vivo mass spectrometry analysis indicated that these peptide contain phosphorylated thr and thr thr and thr are efficiently phosphorylated in vitro by FRAP mTOR when 4E BP1 is bound to eIF4E however phosphorylation at these sites was not associated with loss of eIF4E binding phosphorylated thr and thr are detected in all phosphorylated in vivo 4E BP1 isoforms including those that interact with eIF4E finally mutational analysis demonstrated that phosphorylation of thr thr is required for subsequent phosphorylation of several carboxy terminal serum sensitive sites taken together our results suggest that 4E BP1 phosphorylation by FRAP mTOR on thr and thr is priming event for subsequent phosphorylation of the carboxy terminal serum sensitive sites

We have developed fluorescence based method for mapping single or multiple protein binding sites on straightened large size DNA molecules kbp In the described method protein DNA complexes were straightened and immobilized on flat surface using surface tension fraction of the immobilized complexes displayed sharp DNA bend with two DNA segments extending from the apex the presence of DNA binding protein at the apex was verified by atomic force microscopy the position of protein binding relative to the ends of the DNA molecule was determined by measuring the length of two DNA segments using fluorescence microscopy We demonstrate the potential of the fluorescence based method to localize protein binding sites on the DNA template and to evaluate relative binding affinity the proposed protein binding site mapping technique is simple and easy to perform practical applications include screening for DNA binding protein and the localization of protein binding sites on large segments of DNA

the post genomic era is characterized by the deposition of sequence information for entire genomes in databases currently besides the protein sequence for known human protein there are partial sequence from thousands more human protein for which no biological function has been assigned powerful new tool for the unambiguous identification and characterization of gel separated protein is accomplished by the combination of mass spectrometry and sequence database searching this combination provides the cancer biologist with the ability to identify the potential protein protein associations and ii fully characterize function critical post translational modifications both directly from silver stained polyacrylamide gels In this report we describe the application of tandem mass spectrometry and database searching to two problems which are prototypical for cancer research and indeed for biomedical research in general the first is the identification of gel separated low abundance protein based on amino-acid sequence composition following coimmunoprecipitation with the human apoptosis inhibitor protein BCIX the second is the determination of the precise sites of phosphorylation of the human regulatory protein 4E BP1 which controls mRNA translation

We have determined the relationship between mRNA and protein expression levels for selected gene expressed in the yeast saccharomyces cerevisiae growing at mid log phase the protein contained in total yeast cell lysate were separated by high resolution two dimensional 2D gel electrophoresis over protein spots were excised and identified by capillary liquid chromatography tandem mass spectrometry LC MS MS protein spots were quantified by metabolic labeling and scintillation counting corresponding mRNA levels were calculated from serial analysis of gene expression SAGE frequency tables velculescu zhang zhou vogelstein basrai bassett Jr hieter vogelstein and kinzler cell We found that the correlation between mRNA and protein levels was insufficient to predict protein expression levels from quantitative mRNA data indeed for some gene while the mRNA levels were of the same value the protein levels varied by more than fold conversely invariant steady state levels of certain protein were observed with respective mRNA transcript levels that varied by as much as fold another interesting observation is that codon bias is not predictor of either protein or mRNA levels our results clearly delineate the technical boundaries of current approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient

the generation of cellular ceramides as second messenger has been implicated as regulatory and required step for the induction of apoptosis In this study we have applied recently developed mass spectrometric technique to the determination of changes in physiological ceramide levels during apoptosis induced by tumor necrosis factor plus cycloheximide in U937 cell and the chemical agents anisomycin or geranylgeraniol in HL cell the mass spectrometric method has significant advantages over traditional method for ceramide quantitation in that it determines the relative abundance of all ceramide species present in complex biological lipid mixtures individually and simultaneously We quantitiated ceramides ranging from C14 to C26 finding that their basal levels and relative distribution varied significantly both within and between different cell types however we were not able to detect any significant changes in either total ceramide content or species distribution until or more post stimulation with any of these treatments by which time the cell were in an advanced stage of apoptosis differences were also seen between all three treatments in the ceramide species distribution observed in these late stages of apoptosis these data indicate that in vivo ceramide generation occurs as consequence of apoptosis rather than as an essential second messenger involved in its induction they also pose new questions about the potential roles that certain ceramide species may play in the late stages of apoptosis and demonstrate clear need to utilize the resolving power of mass spectrometry based assay in any future investigations into the biological function of ceramides

mitochondria play key part in the regulation of apoptosis cell death their intermembrane space contains several protein that are liberated through the outer membrane in order to participate in the degradation phase of apoptosis here we report the identification and cloning of an apoptosis inducing factor AIF which is sufficient to induce apoptosis of isolated nuclei AIF is flavoprotein of relative molecular mass which shares homology with the bacterial oxiforeductases it is normally confined to mitochondria but translocates to the nucleus when apoptosis is induced recombinant AIF causes chromatin condensation in isolated nuclei and large scale fragmentation of DNA It induces purified mitochondria to release the apoptogenic protein cytochrome and caspase microinjection of AIF into the cytoplasm of intact cell induces condensation of chromatin dissipation of the mitochondrial transmembrane potential and exposure of phosphatidylserine in the plasma membrane none of these effects is prevented by the wide ranging caspase inhibitor known as VAD fmk overexpression of bcl which controls the opening of mitochondrial permeability transition pores prevents the release of AIF from the mitochondrion but does not affect its apoptogenic activity these results indicate that AIF is mitochondrial effector of apoptotic cell death

the comprehensive analysis of biological system requires combination of genomic and proteomic efforts the large scale application of current genomic technologies provides complete genomic DNA sequence sequence tags for expressed gene EST and quantitative profiles of expressed gene at the mRNA level In contrast protein analytical technology lacks the sensitivity and the sample throughput for the systematic analysis of all the protein expressed by tissue or cell the sensitivity of protein analysis technology is primarily limited by the loss of analytes due to adsorption to surfaces and sample contamination during handling here we summarize our work on the development and use of microfabricated fluidic system for the manipulation of minute amounts of peptide and delivery to an electrospray ionization tandem mass spectrometer new data are also presented that further demonstrate the potential of these novel approaches specifically we describe the use of microfabricated devices as modules to deliver femtomole amounts of protein digests to the mass spectrometer for protein identification We also describe the use of microfabricated module for the generation of solvent gradients at nl min flow rates for gradient chromatography tandem mass spectrometry the use of microfabricated fluidic system reduces the risk of sample contamination and sample loss due to adsorption to wetted surfaces the ability to assemble dedicated modular system and to operate them automatically makes the use of microfabricated system attractive for the sensitive and large scale analysis of protein the comprehensive analysis of biological system requires combination of genomic and proteomic efforts the large scale application of current genomic technologies provides complete genomic DNA sequence sequence tags for expressed gene EST and quantitative profiles of expressed gene at the mRNA level In contrast protein analytical technology lacks the sensitivity and the sample throughput for the systematic analysis of all the protein expressed by tissue or cell the sensitivity of protein analysis technology is primarily limited by the loss of analytes due to adsorption to surfaces and sample contamination during handling here we summarize our work on the development and use of microfabricated fluidic system for the manipulation of minute amounts of peptide and delivery to an electrospray ionization tandem mass spectrometer new data are also presented that further demonstrate the potential of these novel approaches specifically we describe the use of microfabricated devices as modules to deliver femtomole amounts of protein digests to the mass spectrometer for protein identification We also describe the use of microfabricated module for the generation of solvent gradients at nl min flow rates for gradient chromatography tandem mass spectrometry the use of microfabricated fluidic system reduces the risk of sample contamination and sample loss due to adsorption to wetted surfaces the ability to assemble dedicated modular system and to operate them automatically makes the use of microfabricated system attractive for the sensitive and large scale analysis of protein

An injection molded polymeric microfluidic device was coupled to two different types of electrospray ionization ESI mass spectrometers for protein identification We demonstrate that tryptic digests of different protein that were simultaneously present on the polymeric device could be successively mobilized with limited sample to sample cross contamination toward an MS and identified by the collision induced dissociation CID spectra generated from selected peptide

the signal transducer and activator of transcription STAT protein deliver signals from the cell membrane to the nucleus An terminally truncated fragment of murine stat3ß stat3ßtc was produced in bacteria STAT protein must be specifically phosphorylated at single tyrosine residue for dimerization and DNA binding therefore stat3ßtc was coexpressed with the catalytic domain of the elk receptor tyrosine kinase stat3ßtc was quantitatively phosphorylated by this kinase domain gel filtration chromatography revealed stat3ßtc dimer Y705 was identified as the major phosphorylated residue of stat3ßtc this corresponds to the tyrosine residue which is phosphorylated by the janus kinase in vivo the phosphorylated stat3ßtc specifically bound to DNA binding sites the described protocol allows the production of large amounts of activated protein for biochemical and pharmaceutical studies copyright federation of european biochemical societies

one of the most dramatic current developments in bio logical research is the shift from the analysis of single gene and protein to the comprehensive analysis of bi ological system and pathways this shift is at least in part the consequence of the development of automated high throughput genomic technologies which have advanced to point where in principle it has become pos sible to determine complete genome sequence and to quantitatively measure the mRN levels of all the gene expressed in cell currently no comparably powerful technology is available for the analysis of biological sys tems on the protein level protein are however the most significant class of biological effector molecules and complete model of biological process cannot be established without knowledge of the identity function and state of expression and activity of the protein in volved quantitative expression map of the protein in cell or tissue has been termed proteome We will describe suite of technologies for the identification and analysis of the protein which constitute biological sys tem for the description of proteomes mass spectrometry based identification of protein numerous techniques for the identification of pro teins have been described and successfully employed they include terminal and internal protein sequenc ing identification by amino-acid composition analysis and variety of mass spectrometric MS approaches currently MS or tandem mass spectrometric MS MS analysis of protein digest and correlation of the obtained data with sequence database entries is considered the method of choice for protein identification the sensitivity and the throughput of these techniques are in versely related the most sensitive techniques such as solid phase extraction capillary electrophoresis MS MS and nanospray MS MS have been difficult to automate techniques of lower sensitivity such as liquid chroma tography MS MS have successfully been automated for higher sample throughput To achieve both high sensi tivity and automation in the same system we have used photolithography etching techniques to develop micro machined devices which can be connected on line with electrospray ionization ESI MS MS multiple samples concurrently present in different reservoirs on the device are sequentially and automatically mobilized by an array of high voltage relays and electroosmotically pumped either directly or after chromatographie or electrophor etic separation to the ion source of the MS instrument We have used this system for the identification of nu merous protein separated by high resolution two dimensional gel electophoresis analysis of the state of protein modification frequently the state of modification of protein and the specific constellation of modified protein in cell indicate the state of activity of the protein and the cell respectively the analysis of protein modifications is complicated by frequently low stoichiometry and complex patterns of modification low abundance of the modified protein and the large number and chemical diversity of protein modifications the task of analyzing the state of protein modification in biological system has at least three stages the first is the detec tion visualization of the modified protein in protein mixture the second step is the identification of those protein in protein mixture which are modified by specific modification the third step is the localization of the modification within the polypeptide chain of the identified protein protein and peptide MS and MS MS are rapidly becoming the method of choice for the identification as well as the analysis of posttransla tional modifications We have developed and successfully applied suite of techniques for the analysis of the phosphorylation state of protein reversible phosphorylation of protein at specific sites is an essentially universal mechanism for the control of the activity of the phosphorylated protein and of biological process the objectives of the techniques developed were the quantitative determination of the state of phosphorylation of the small amounts of phosphoprotein which can be isolated from cell or tis sues representing specific state these objectives were achieved by combination of biochemical enzymatic chromatographie and electrophoretic techniques which were used together with ESI MS MS the identification of the protein in complex pro tein mixture which are modified by specific type of modification is almost universally achieved by metabolic radiolabeling of the protein sample with metabolic precursor specific for the modification under investigation followed by the separation of the protein mixture by high resolution gel electrophoresis and detection of the modified protein by autoradiography fluorography or storage phosphorimaging this technique is purely de scriptive and does not in general yield the identity of the modified protein We will describe novel approaches based on ESI MS MS which in the same operation detect the protein modified by specific group in complex protein mixtures and also identify the protein by its amino-acid sequence construction of multidimensional protein expression maps with current technology global and quantitative mRNA transcript expression maps can be established rapidly the construction of similarly comprehensive protein expression maps proteome maps is much more labor intensive and much slower the added cost time and effort required to establish proteome maps is justified by the added information regarding the state of biological system which can be obtained from proteome analysis We have correlated the quantitative mRNA transcript expression map obtained from the yeast saccharomyces cerevisiae with quantitative proteome map from the same strain of yeast grown under identical conditions the quantitative mRNA expression data were calculated from SAGE frequency tables described in the literature velculescu et al the protein expression data were derived from the quantitative and mass spectrometric analysis of homogeneous protein spots in two dimensional gels the gels were generated by separating total yeast cell lysates metabolically radiolabeled to equilibrium the analysis of the results indicated that the mRNA transcript and protein expression levels for specific gene are poorly correlated ii protein coded for by the same gene frequently migrated to different positions in the two dimensional protein pattern indicating the activity of posttranslational modification and processing mechanisms and iii without selective enrichment for low abundance protein current proteome technology is limited to the analysis of the most highly expressed protein in cell and is therefore not comprehensive We therefore conclude that efforts in proteome analysis should be focused on the determina tion of those parameters describing the state of biological system which cannot be determined by genomic or genetic means such parameters include the quantity of protein expression protein half life the state of modification of individual protein and the state of association of protein with other molecules protein analysis technology is in the process of being transformed from technology focusing on the analysis of single protein to the analysis of all the protein which constitute biological system MS and MS MS have become the method of choice for both the identification of protein in biological system and the analysis of their covalent structure the limitations inherentin current protein analytical technologies with respect to sensitivity and sample throughput suggest that protein analysis is best practiced in conjunction with genomic analysis and that protein analysis should focus on the determination of those parameters characterizing biological system which can only be determined by direct protein analysis

We describe simple solvent delivery system for gradient capillary HPLC at nanoliter per minute flow rates the novel aspect of the system is that solvents are delivered one at time using switching valve into relatively large volume mixing chamber efficient mixing in the chamber causes the formation of sigmoidal gradient from the initial solvent to the subsequent solvent which is then delivered to capillary column the shape of the gradients formed can be predicted from simple theoretical model gradients of different slope can be formed by varying either the size of the chamber or the system flow rate the system is robust reproducible and simple to operate We provide detailed protocol of how to construct low cost capillary HPLC system consisting of two syringe pumps capillary mixing chamber capillary column and zero dead volume microelectrospray interface We demonstrate that the coupling of this HPLC system to mass spectrometer enabled us to identify protein at the low femtomole level in solution phase digests and at the picomole level in digests of samples separated on SDS PAGE gels We believe that the strategy presented will be useful as general method for the characterization of protein and peptide by capillary HPLC electrospray mass spectrometry

ß3 hexosaminidase hex is an essential lysosomal enzyme whose activity is higher in the epididymis than in other tissues the enzyme is also present in sperm and has been postulated to be required for fertilization To better understand the role of hex in reproduction we have examined the testes and epididymides of mouse models of human tay sachs and sandhoff diseases produced by targeted disruption of the hexa subunit or hexb subunit gene respectively encoding the enzymes hex structure aß and hex ßß testis weight morphology and sperm counts were unaffected in hex deficient mice In the epididymis of the hex deficient hexa mice there was large increase in the size and number of lysosomes in the initial segment intermediate zone In hexb mice hex and deficient the epididymal defects were much more extensive and the cytoplasm of all cell types throughout the efferent ducts and epididymis was filled with pale uncondensed enlarged lysosomes In contrast to the brain where M2 ganglioside accumulates both mutaut mice accumulated two non M2 gangliosides in the epididymis the major accumulated species was characterized by electrospray ionization tandem mass spectrometry the hexa male mice were fertile however litter sizes were reduced the hexb males were able to sire normal sized litters up to nine weeks of age and remained healthy until weeks of age the extensive abnormalities in the hexb mice in contrast to region specific effects in the hexa mice indicate an important and novel role for the hex isozyme in the epididymis and region specific role for hex in the initial segment intermediate zone In contrast to other reports our results indicate that hex is not essential for fertilization in young adult male mice To explain the extensive epididymal abnormalities in the hexb mice we propose that substrates for hex such as testis derived glycolipids cannot be catabolized and accumulate in lysosomes leading to epididymal dysfunction and abnormalities in the epididymal luminal environment that supports sperm maturation

major histocompatibility class II MHC II molecules are transmembrane protein that have central role in development and control of the immune system they are encoded by multigene family and their expression is tightly regulated MHC II deficiency OMIM is an autosomal recessive immunodeficiency syndrome resulting from defects in trans acting factors essential for transcription of MHC II gene there are four genetic complementation groups and reflecting the existence of four MHC II regulators the factors defective in groups CIITA RFX5 and RFXAP have been identified CIITA is non DNA binding co activator that controls the cell type specificity and inducibility of MHC II expression RFX5 and RFXAP are two subunits of RFX multi protein complex that binds the box motif of MHC II promoters mutations in the gene encoding RFX5 RFX5 or RFXAP RFXAP abolish binding of RFX refs similar to groups and group is characterized by defect in RFX binding and although it accounts for the majority of patients the factor defective in group has remained unknown We report here the isolation of RFX by novel single step DNA affinity purification approach and the identification of RFXANK the gene encoding third subunit of RFX RFXANK restores MHC II expression in cell lines from patients in group and is mutated in these patients RFXANK contains protein protein interaction region consisting of three ankyrin repeats its interaction with RFX5 and RFXAP is essential for binding of the RFX complex to MHC II promoters

We have developed an atomic force microscopy AFM based method for mapping protein binding sites on individual long DNA molecules kb at nanometer resolution the protein is clearly detected at the apex of the bent DNA molecules randomly coiled DNA molecules or protein DNA complexes were extended by motor controlled moving meniscus on an atomically flat surface the immobilized molecules were detected by AFM the straightened DNA displayed sharp bend at the site of bound protein with the two DNA segments linearly extending from the protein binding site using GAL4 yeast transcription factor we demonstrate good agreement of the position of the observed binding site on straightened DNA templates to the predicted binding site the technique is expected to have significant implications in elucidating DNA and protein interactions in general and specifically for the measurement of promoter occupancy with unlabeled regulatory protein at the single molecule level

We describe the coupling of microfabricated fluidic device to an electrospray ionization ESI quadrupole time of flight mass spectrometer QqTOFMS for the identification of protein samples the microfabricated devices consisted of three reservoirs connected via channels to main capillary which in turn was linked via microspray interface to the QqTOFMS here we present preliminary results obtained using this system standardized solutions of myoglobin tryptic digest were analyzed indicating limit of detection at the low to sub fmol µL the combination of the microfabricated device for rapid sample delivery and the rapid acquisition capability enhanced resolution and mass accuracy of the QqTOF offers unique possibilities for the rapid identification of protein by database searching this platform can generate MS data suitable for protein database searching by the peptide mass fingerprinting approach and MS MS data suitable for protein database searching here the results of the two database searching approaches are compared and the possibilities of combining the two approaches for rapid identification of protein are discussed also we present comparison of the results obtained using the three position microfabricated device coupled to the ESI QqTOFMS and to an ESI ion trap MS finally the combination of terminal 18O labeling of peptide and the microfabricated system for automated combined peptide mass fingerprinting and sequence tag database searching is discussed

We cloned the cDNA for human RGSZ1 the major selective GTpase activating protein GAP in brain wang Tu woodson song and ross biol chem and member of the RGS family of protein GAPs its sequence is identical to RET RGS1 except its terminal extension and identical to GAIP purified recombinant RGSZ1 RET RGS1 and GAIP each accelerated the hydrolysis of Ga GTP over fold with values of nM RGSZ1 was fold selective for Ga over Ga1 unusually specific among RGS protein other enzymological properties of RGSZ1 brain GAP and RET RGS1 were identical GAIP differed only in Mg2+ dependence and in its slightly lower selectivity for Ga RGSZ1 RET RGS1 and GAIP thus define subfamily of GAPs within the RGS protein RGSZ1 has no obvious membrane spanning region but is tightly membrane bound in brain its regulatory activity in membranes depends on stable bilayer association when co reconstituted into phospholipid vesicles with and m2 muscarinic receptors RGSZ1 increased agonist stimulated GTpase gt fold with EC50 lt nM but RGSZ1 added to the vesicle suspension was lt as active RGSZ1 RET RGS1 and GAIP share cysteine string sequence perhaps targeting them to secretory vesicles and allowing them to participate in the proposed control of secretion by phosphorylation of Ga by protein kinase inhibited the GAP activity of RGSZ1 and other RGS protein providing mechanism for potentiation of signaling by protein kinase

We have previously described the use of solid phase extraction SPE capillary zone electrophoresis CZE tandem mass spectrometry MS MS system for protein analysis at the low femtomole to subfemtomole level here we describe the systematic optimization of number of parameters which facilitate the use of the SPE CZE MS MS system and further enhance its performance specifically we describe robust SPE cartridge design which can be assembled without the use of glue the evaluation of procedures to chemically modify the inner wall of the fused silica capillaries used in the system to improve separation and reproducibility and the comparison of different reverse phase RP resins used for the SPE cartridge We also explored the effects of transient isotachophoresis with respect to system performance and compatibility with different fused silica surface coatings the RP resins used and MS MS the enhanced performance of the optimized system is demonstrated by the analysis of calibrated tryptic digests of bovine serum albumin BSA

We describe an integrated analytical system consisting of microfluidics device micromachined using photolithography etching technology panel of computer controlled high voltage relays and an electrospray ionization tandem mass spectrometer movement of solvents and samples on the device and off the device to the mass spectrometer was achieved by directed electroosmotic pumping induced by the activation of suitable constellation of high voltage relays the system was used for the sequential automated analysis of protein digests We demonstrate low femtomole per microliter sensitivity of detection and compatibility of the system with the automated analysis of protein separated by two dimensional gel electrophoresis

microfabrication technology offers the opportunity to construct microfluidic modules which are designed to perform specific dedicated functions here we report the construction of microfabricated device for the generation and delivery by electroosmotic pumping of solvent gradients at nanoliter per minute flow rates the device consists of three solvent reservoirs and channels which were etched in glass solvent gradients and solvent flows were generated by computer controlled differential electroosmotic pumping of aqueous and organic phase respectively from the solvent reservoirs the device was integrated into an analytical system consisting of the solvent gradient delivery module reverse phase microcolumn and an electrospray ionization ion trap mass spectrometer MS the system was used for the analysis at high sensitivity of peptide and peptide mixtures generated by proteolytic digestion of protein We have measured an absolute limit of detection as low as fmol and concentration limit of detection at the amol µL level the system was also successfully used for the identification of protein separated by 1D and 2D gel electrophoresis this was achieved by gradient frontal analysis of die peptide mixture generated by proteolysis of the respective protein and the automated generation and interpretation of collision induced dissociation spectra

We discuss how structural domain boundaries liquid drop boundaries and other topologically one dimensional features can be used to focus or to define the spatial extent of deposition or chemical reaction We also show how reactants can be delivered specifically to such boundaries using novel deposition strategies such strategies potentially provide simple route to create tailored stable nanometre scale structures

In this review we examine the current state of proteome analysis there are three main issues discussed why it is necessary to study proteomes how proteomes can be analyzed with current technology and how proteome analysis can be used to enhance biological research We conclude that proteome analysis is an essential tool in the understanding of regulated biological system current technology while still mostly limited to the more abundant protein enables the use of proteome analysis both to establish databases of protein present and to perform biological assay involving measurement of multiple variables We believe that the utility of proteome analysis in future biological research will continue to be enhanced by further improvements in analytical technology

analytical and preparative electrophoresis separation techniques have been essential tools in protein biochemistry and the biological sciences in general the combination of high resolution electrophoresis techniques with high performance analytical procedures has dramatically enhanced analytical protein biochemistry In this report we describe the combination of electrophoretic separation techniques with electrospray ionization ESI tandem mass spectrometry MS MS series of different techniques consisting of automated high performance liquid chromatography HPLC MS MS capillary HPLC MS MS and solid phase extraction SPE capillary zone electrophoresis CZE MS MS are described in the context of the identification of high pmol to the low fmol amounts of protein application of these powerful new tools for the analysis of protein on large proteome wide scale is presented furthermore the combination of orthogonal separation techniques such as immobilized metal affinity chromatography IMAC with SPE CZE MS MS and IMAC followed by HPLC and by SPE CZE MS MS are presented for the detailed investigation of post translational modifications of specific protein

analytical biochemistry in particular the analysis of regulatory protein that control biological system and pathways is dependent on method of ever increasing sensitivity capillary electrophoresis CE has long been recognized as an ultrasensitive analytical technique In spite of the high sensitivity CE has not penetrated protein discovery research as standard analytical method In this review article we summarize recent technical developments which have significantly enhanced CE as tool for the analysis of trace amounts of protein specifically we review recent advances in the development and application of capillary electrophoresis mass spectrometry CE MS and on line analyte concentration techniques and introduce the emerging field of microfluidics as front end to mass spectrometry MS

We have compared several different experimental system currently in use in our laboratory for protein identification by high performance liquid chromatography electrospray ionization tandem mass spectrometry HPLC ESI MS MS after sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE the efficiency of peptide recovery from trypsin digested gel bands or electroblotted membrane slices was examined using 35S labeled yeast protein and was found to be in excess of dilution series of two standard protein bovine serum albumin BSA and carbonic anhydrase CA was analyzed by HPLC ESI MS MS to determine what amount of protein could be loaded onto gel and successfully identified measure we refer to as the practical detection limit We were able to identify both standards at the ng level in samples prepared from gel slices using either regular spray or flow split microspray HPLC MS interface system In samples prepared from membrane pieces carbonic anhydrase was also identified at the ng level while bovine serum albumin could only be identified in samples of more than ng In general protein identification was slightly better in samples prepared from gels rather than membranes dilution series of lesser amounts of the same standard protein was also analyzed using gradient capillary LC system and we were able to successfully identify ng of carbonic anhydrase and ng of BSA

In birds intestinally derived lipoproteins are thought to be secreted directly into the portal vein rather than to enter the circulation via the lymphatic system as in mammals hepatic clearance of these so called portomicrons must be rapid but the protein mediating their catabolism presumably analogues of the kDa mammalian apolipoprotein have not been identified In searching for such mediator we have isolated hitherto unknown kDa protein from chicken serum which we identified by microsequencing and molecular cloning as counterpart to mammalian apolipoprotein AIV apoAIV mature chicken apoAIV consists of amino-acid lacks cysteine residue and displays sequence identity with human apoAIV and to significantly lesser extent with apoAIVs of rodents this first nonmammalian apoAIV characterized is the smallest homologue reported so far because of the lack of repeated motifs at the carboxyl terminus with the consensus sequence glu gln glu ala gln hallmark of mammalian apoAIVs chicken apoAIV isoelectric point is also considerably more acidic than its human counterpart agarose gel electrophoresis revealed that unlike human apoAIV which migrates to pre position chicken apoAIV shows fast migration functional characterization demonstrated that the avian protein is able to activate the enzyme lecithin cholesterol acyltransferase roosters and hens express apoAIV predominantly in the gut one fifth as much in the liver and no other sites of expression are identifiable by northern blot analysis although pronounced intestinal synthesis is common to apoAIVs the features of the avian protein support the notion that it represents prototype of an apoprotein that evolved to acquire possibly distinct functions in mammals and birds

We describe an integrated workstation for the automated high throughput and conclusive identification of protein by reverse phase chromatography electrospray ionization tandem mass spectrometry the instrumentation consists of refrigerated autosampler submicrobore reverse phase liquid chromatograph and an electrospray triple quadrupole mass spectrometer for protein identification enzymatic digests of either homogeneous polypeptides or simple protein mixtures were generated and loaded into the autosampler samples were sequentially injected every min ions of eluting peptide were automatically selected by the mass spectrometer and subjected to collision induced dissociation following each run the resulting tandem mass spectra were automatically analyzed by SEQUEST program that correlate uninterpreted peptide fragmentation patterns with amino-acid sequence contained in databases protein identification was established by SEQUEST SUMMARY program that combines the SEQUEST scores of peptide originating from the same protein and ranks the cumulative results in short summary the workstation performance was demonstrated by the unattended identification of protein from the yeast saccharomyces cerevisiae which were separated by high resolution two dimensional PAGE the system was found to be very robust and identification was reliably and conclusively established for protein if quantities exceeding pmol were applied to the gel the level of automation the throughput and the reliability of the results suggest that this system will be useful for the many projects that require the characterization of large numbers of protein

A panel of powerful genomic technologies now permits the establishment of profil the expressed gene in cell dependent on parameters such as the state tifferentiation or state of activation for the comprehensive analysis of biologicy process it is essential that both the level of expression and the state of activaty of th 3roteins mediating specific functions are also systematically investigated the level tctivity of protein is frequently controlled by post translational editing events such 9roteolytic processing group specific modification and complex formation with sma igands or other bio polymers We have developed technology for the establishment of protein expression profiles ells and for the identification and localization of regulatory modifications within th polypeptide chain the approach is based on high resolution two dimensional electrophoresis for the separation Of complex protein mixtures and on electrospra onization tandem mass spectrometry for the primary structural analysis of the separate protein We will describe integrated analytical system which combine on line tl high resolution peptide separation techniques capillary chromatography or capillal electrophoresis with tandem mass spectrometry these system were used for tl automated high throughput identification of protein separated by 2DE and for tldetermination of the state of modification of protein We demonstrate that tl sensitivity level of the system is at par with or exceeds the sensitivity the most sensitive protein staining method We discuss applications of this technology to the dissectic of regulatory pathways in eukaryotic cell which are controlled by reversible protein phosphorylation

We describe the separation and detection at the low femtomole level of 3pyridinylmethylaminocarboxypropyl phenylthiohydantoins PTHs by capillary liquid chromatography microelectrospray ion trap mass spectrometry highest sensitivity was obtained in the multiple ion monitoring operating mode in which we detected PTHs at the fmol level with signal to noise ratio of approximately We investigated the fragmentation patterns of the isobaric PTH isoleucine and PTH leucine by electrospray ionization ion trap tandem mass spectrometry the compounds could be differentiated by fragment ion of mass which was specific for the breakdown of PTH leucine thus allowing for the unambiguous identification of the PTH derivatives of all naturally occurring amino-acid by their masses and fragmentation patterns

the fas receptor is one of number of important physiological inducers of programmed cell death apoptosis current models for regulation of this process involve rapid conversion of sphingomyelin to ceramide by cellular sphingomyelinases induced changes in cellular levels of such sphingosine based ceramides are normally extrapolated from measurements of sphingomyelinase activity or following their conversion to ceramide phosphate via treatment of cellular lipid extracts with bacterial diacylglycerol kinase DAGK To allow direct study of cellular sphingosine and sphinganine based ceramide levels we developed mass spectrometric technique capable of determining inducible changes in both overall ceramide levels and species distribution in cellular lipid preparations contrary to current models we detected no changes in cellular ceramide levels up to hours post stimulation of jurkat cell with an anti fas IgM even though this treatment did induce apoptosis We also determined in the same system that increases in cellular ceramide levels assessed by DAGK assay were due to enhancement of AGK activity rather than ceramide as substrate thus the first direct measurement of ceramides present in cell undergoing apoptosis indicate that the induction of apoptosis does not involve sphingosine based ceramides as second messengers contrary to many published accounts

An anti axolemma monoclonal antibody designated G21 has been isolated in order to understand molecular mechanisms involved in myelination both biochemical and morphological studies showed that the monoclonal antibody inhibits myelin production by oligodendrocytes in cerebellar slice cultures On western blots of axolemma preparations the antibody recognized and kD protein the present study involves the isolation and characterization of the G21 antigen the G21 immunoreactive protein of and kD were purified from the adult rat sciatic nerve and amino-acid sequencing of these protein revealed significant homology to aI and aII chains of collagen type biochemical and western blot analysis using pure collagen collagen antibody and collagenase suggest that the antigen isolated from sciatic nerve is collagen however immunofluorescence studies using the G21 antibody collagen antibody collagenase and northern blot analysis using collagen probe do not fully support the view that the G21 antigen in the CNS is also collagen We conclude that the G21 antigen is collagen like protein involved in CNS myelination

T lineage specific activation antigen TLiSA1 antigen was initially described as lineage specific activation antigen involved in the differentiation of human cytotoxic cell subsequently the antigen was identified on platelets and was shown to be involved in platelet activation hence it was renamed platelet and cell antigen PTA1 although identity between the two antigens was not established In the present study we have cloned the cDNA encoding TLiSA1 from jurkat cell and show it to be novel member of the immunoglobulin superfamily with the unusual structure of two domains only identity between TLiSA1 and platelet PTA1 is established by immunological criteria by internal peptide sequence obtained from the purified platelet glycoprotein and by sequencing the platelet transcript after reverse transcriptase polymerase chain reaction In jurkat cell TLiSA1 PTA1 mRNA and surface protein expression is greatly stimulated by treatment of the cell with phorbol ester but the cell proliferative signal of phorbol ester and ionophore combined greatly reduces or abrogates this response and this suppressive effect of the ionophore is not reversed by incorporating FK506 to inhibit calcineurin together with the known signaling role of PTA1 these data substantiate the notion that this molecule is implicated in cell differentiation perhaps by engagement of an adhesive ligand

nanoelectrospray mass spectrometry the infusion at low flow rates of unseparated peptide mixtures representing protein proteolytic digests into an electrospray ionization mass spectrometer MS has been shown to be suitable method for the analysis of small amounts of protein however the current technique is time consuming tedious and difficult to automate We used microfabrication technologies to construct device for the sequential infusion of different peptide samples into an electrospray ionization MS without the need for sample manipulation In this device etched sample and buffer reservoirs are connected via etched channels to microelectrospray ion source peptide samples typically unseparated tryptic digests of protein are applied to different reservoirs flow of liquid originating from specific reservoir is generated and selectively directed toward the microsprayer and the MS by electroosmotic pumping the analyte protein are identified by searching sequence databases with the information contained in the collision induced spectra of selected peptide with this system we have achieved limit of detection in the low femtomoles per microliter range for peptide standards We also show that samples deposited in different reservoirs can be sequentially mobilized without cross contamination and that protein can be conclusively identified at the low femtomoles per microliter level the successful coupling online of microfabricated devices to an electrospray ionization MS represents an essential step toward the construction of automated high throughput and high sensitivity analytical system

We describe the separation and detection at the low femtomole level of pyridinylmethylaminocarboxypropyl phenylthiohydantoins PTHs by capillary liquid chromatography microelectrospray ion trap mass spectrometry highest sensitivity was obtained in the multiple ion monitoring operating mode in which we detected PTHs at the fmol level with signal to noise ratio of approximately We investigated the fragmentation patterns of the isobaric PTH isoleucine and PTH leucine by electrospray ionization ion trap tandem mass spectrometry the compounds could be differentiated by fragment ion of mass which was specific for the breakdown of PTH leucine thus allowing for the unambiguous identification of the PTH derivatives of all naturally occurring amino-acid by their masses and fragmentation patterns

two procedures for the derivatization of the inner wall of fused silica capillaries for the analysis of peptide and protein by capillary electrophoresis CE at neutral pH are presented In the first procedure polyethyleneimine PEI is covalently attached to the capillary wail In the second procedure PEI is additionally cross linked We present analysis of standard peptide and protein by CE using the coated capillaries these coatings will have application for the separation of protein complexes at neutral pH prior to analysis by electrospray mass spectrometry

the fas receptor is one of number of important physiological inducers of programmed cell death apoptosis current models for regulation of this process involve rapid conversion of sphingomyelin to ceramide by cellular sphingomyelinases induced changes in cellular levels of such sphingosine based ceramides are normally extrapolated from measurements of sphingomyelinase activity or following their conversion to ceramide phosphate by treatment of cellular lipid extracts with bacterial diacylglycerol kinase DAGK To allow direct study of cellular sphingosine and sphinganine based ceramide levels we developed mass spectrometric technique capable of determining inducible changes in both overall ceramide levels and species distribution in cellular lipid preparations contrary to current models we detected no changes in cellular ceramide levels up to hr poststimulation of jurkat cell with an anti fas IgM although this treatment did induced apoptosis We also determine in the same system that when utilizing the DAGK assay increased phosphorylation of substrates that comigrated with ceramide standards was apparent but that this effect was due to an enhancement of DAGK activity rather than increases in levels of cellular ceramides as substrates per se thus the first direct measurement of ceramides present in cell undergoing apoptosis indicates that insofar as it can be measured the induction of apoptosis does not involve the generation of sphingosine based ceramides contrary to many published accounts

stimulation of the cell antigen receptor TCR activates set of non receptor protein tyrosine kinases that assist in delivering signals to the cell interior among the presumed substrates for these kinases adaptor protein which juxtapose effector enzyme system with the antigen receptor complex figure prominently previous studies suggested that lnk kDa protein consisting of single SH2 domain and region containing potential tyrosine phosphorylation sites might serve to join grb2 phospholipase and phosphatidylinositol kinase to the TCR To elucidate the physiological roles of lnk in cell signal transduction we isolated the mouse lnk cDNA characterized the structure of the mouse lnk gene and generated transgenic mice that overproduce lnk in thymocytes here we report that although lnk becomes phosphorylated during cell activation it plays no limiting role in the TCR signaling process moreover we have distinguished p38 lnk from the more prominent kDa tyrosine phosphoproteins that appear in activated cell together these studies suggest that lnk participates in signaling from receptors other than antigen receptors in lymphocytes

mass spectrometric techniques for the identification of protein either by amino-acid sequencing or by correlation of mass spectral data with sequence databases are becoming increasingly sensitive and are rapidly approaching the limit of detection achieved by the staining of protein in gels or after electroblotting on membranes here we present technique for the sensitive staining of protein electroblotted onto nitrocellulose or polyvinylidene difluoride membranes and enzymatic cleavage conditions for such protein to achieve optimal recovery of peptide the technique is based on the deposition of colloidal silver on the membrane bound protein peptide mixtures generated by proteolysis on the membrane were recovered at high yields and were compatible with analysis by reverse phase chromatography and on line electrospray ionization mass spectrometry this simple and rapid colloidal silver staining procedure allowed the visualization of less than ng of protein in band and thus approached the sensitivity of silver staining in gels We demonstrate that this method allows the detection of subpicomole amounts of electroblotted protein and their identification by high performance liquid chromatography electrospray ionization tandem mass spectrometry

pim is an oncogene encoded serine threonine kinase expressed primarily in cell of the hematopoietic and germ line lineages previously identified only in mammals pim cDNA was cloned and sequenced from the african clawed frog xenopus laevis the coding region of xenopus pim encoded protein of residue which exhibited amino-acid identity with the full length human cognate xenopus pim was expressed in bacteria as glutathione transferase GST fusion protein and in COS cell phosphoamino acid analysis revealed that recombinant pim autophosphorylated on serine and threonine and to more limited extent on tyrosine electrospray ionization mass spectroscopy was undertaken to locate these phosphorylation sites and the primary autophosphorylation site of GST pim was identified as set with thr and set being minor sites set which immediately follows the high conserved asp phe gly motif in catalytic subdomain VII is also featured in more than other protein kinases To evaluate the importance of the ser site on the phosphotransferase activity of pim ser was mutated to either alanine or glutamic acid and the constructs were expressed in bacteria as GST fusion protein and in COS cell these mutants confirmed that ser is major autophosphorylation site of pim and indicated that phosphorylation of pim on the ser residue may serve to activate this kinase

A kDa protein present in the glucose transporter GLUT4 isotype containing vesicles from rat adipocytes has been isolated the sequence of two tryptic peptide were obtained and on the basis of these its cDNA partially cloned the kDa protein is almost certainly identical to major integral glycoprotein of this size in the rat adipocyte plasma membrane since its predicted terminal sequence is the same as that recently determined for this glycoprotein by amine acid sequencing moreover the predicted partial sequence amino-acid of the kDa protein is highly homologous to the corresponding region of human placental amine oxidase which was cloned simultaneously and proposed to be secreted protein the amino-acid sequence of the kDa rat human amine oxidase indicates that the protein consists of very short terminal cytoplasmic domain followed by single transmembrane segment and large extracellular domain containing the catalytic site thus this study establishes the kDa rat human amine oxidase as the first integral membrane amine oxidase to be cloned the membrane amine oxidase was more abundant in the plasma membranes than the low density microsomes of the adipocyte and in contrast to some other protein found in GLUT4 vesicles it did not redistribute to the plasma membrane in response to treatment of the cell with insulin

CTP phosphocholine cytidylyltransferase CT the rate controlling enzyme in phosphatidylcholine biosynthesis is activated by reversible membrane binding To investigate the membrane binding mechanism of CT we have used the photoreactive hydrophobic probe trifluoromethyl 125I iodophenyl diazirine 125I TID association of CT with phosphatidylcholine oleic acid vesicles was first demonstrated by gel filtration analysis upon irradiation CT was covalently labeled by 125I TID presented in phosphatidylcholine oleic acid vesicles this demonstrates an intercalation of part of the protein into the hydrophobic core of the membrane To identify the membrane embedded domain the chymotrypsin digestion products of 125I TID labeled CT were analysed chymotrypsin digestion produced set of previously defined terminal fragments craig johnson and cornell biol chem as well as several small terminal fragments which react with an anti peptide antibody raised against the proposed amphipathic helix all fragments containing the amphipathic helical region of the enzyme had 125I TID label associated while the chymotryptic fragment which lacked this region was not highly labeled similar fragment labeling patterns were produced when 125I TID was presented in phosphatidylcholine oleic acid or phosphatidylcholine diacylglycerol vesicles suggesting that the same domain of CT mediates binding to membranes containing either of the two lipid activators residue synthetic peptide corresponding in sequence to the amphipathic helical region of CT was labeled with 125I TID demonstrating its ability to intercalate independently of the rest of the protein these results indicate membrane binding mechanism for cytidylyltransferase involving the intercalation of the amphipathic helix region into the hydrophobic acyl chain core of the activating membrane

recently we have shown that solid phase microextraction capillary electrophoresis device coupled to an electrospray ionization triple quadrupole mass spectrometer through microelectrospray interface represents powerful analytical system for the rapid conclusive and sensitive identification of protein separated by gel electrophoresis here we report on the successful coupling of the same device to an electrospray ion ization ion trap mass spectrometer and on the comparative evaluation of the performance of the triple quadrupole and ion trap based system In the ion trap mass spectrometer based system using tryptic digest of calibrated bovine serum albumin sample we achieved limits of detection in the single mass spectrometry MS and tandem MS mode respectively of amol if µL of solution at concentration of amol µL was applied the system was also successfully used to identify six yeast protein isolated from single analytical two dimensional polyacrylamide gel for the detection of unfragmented peptide ions both system showed comparable sensitivity whereas the ion trap based system showed superior performance with fragment ion spectra

capillary electrophoresis tandem mass spectrometry has been used successfully for the analysis of complex peptide mixtures the method is limited by relatively high concentration limit of detection and by matrix effects here we describe on line coupling of solid phase microextraction device to capillary electrophoresis tandem mass spectrometry system the performance of the integrated instrument was evaluated for the identification of protein by their amino-acid sequence We report that the concentration limit of detection was improved at least fold to the low attomole µl range and that matrix effects were minimized by extensive sample clean up during solid phase extraction We demonstrate that the implementation of solid phase extraction device significantly enhances capillary electrophoresis tandem mass spectrometry as method for the identification of low abundance protein isolated from high resolution two dimensional polyacrylamide gels

ceramides and sphingoid bases are important intracellular second messengers that play role in the regulation of cell growth differentiation and programmed cell death until now quantitative and qualitative analysis of ceramide second messengers has been limited by lack of analytical method capable of detecting endogenous levels of and differentiating between individual ceramide species here we report the use of electrospray ionization tandem mass spectrometry for the qualitative and quantitative analysis of ceramides collision induced fragmentation resulted in characteristic product ions for the sphingosine and dihydrosphingosine sphinganine head groups at and and and respectively regardless of the length of the fatty acyl chains with spectra being reproducible at concentrations as low as nM fmol µl these reporter ions were used to detect both sphingosine and sphinganine based ceramides in complex mixtures using precursor ion scan analysis We demonstrated the application of this method for profiling the composition of ceramides in commercial preparation of bovine brain ceramides and following minimal chromatographic separation lipid extract of cultured cell furthermore we easily detected relative differences in individual ceramide species levels by comparing the profiles of three related lymphocyte cell lines jurkat U937 and WEHI finally by the addition of nonnaturally occurring internal standard we show that the technique can be used to measure quantitative changes in ceramide levels in such biologically derived lipid preparations

polypeptides from the PS holocomplex of the red alga cruentum purified for microsequencing confirmed that six LHC polypeptides from SDS PAGE are distinct apoproteins analysis of cDNA clone designated as lhcaR2 from cDNA library indicates that it shares major structural features with the recently cloned first red algal gene lhcaR1 the lhcaR2 is believed to encode the kDa polypeptide of the LHC complex from comparison of the deduced amino-acid sequence and the microsequences of several tryptic digest fragments from the isolated polypeptide As in chlorophytic and chromophytic LHCs the essential residue for chl binding and helix stabilization in helices and are highly conserved relatedness between rhodophytes and the chlorophytes is also inferred from sequence conservation in the flanking regions of helices and conversely helix exhibited the highest similarity between LHC sequence of chl binding chromophytes and the chl binding rhodophytes with of residue identical or conservatively substituted moreover whereas in chlorophytes the and chl binding residue are separated by seven amino-acid residue they are always separated by residue in rhodophytes and chromophytes superimposition of the predicted lhcaR2 sequence with the LHC II model kuhlbrandt et al nature shows the same structural features except shortened connecting sequence between helices and on the lumenal side the chimeric nature of rhodophyte gene with both chromophytic and chlorophytic features leads to the suggestion that they reflect attributes of an intermediate stage in LHC apoprotein evolution

the prochlorophytes are oxygenic prokaryotes differing from other cyanobacteria by the presence of light harvesting system containing both chlorophylls chls and and by the absence of phycobilins We demonstrate here that the chl binding protein from all three known prochlorophyte genera are closely related to isiA cyanobacterial chl binding protein induced by iron starvation and to CP43 constitutively expressed chl antenna protein of photosystem II the prochlorophyte chl protein pcb gene do not belong to the extended gene family encoding eukaryotic chl and chl light harvesting protein although higher plants and prochlorophytes share common pigment complements their light harvesting system have evolved independently

ZAP is protein tyrosine kinase PTK that plays critical role in cell activation To study the role of ZAP catalytic activity in this process substrate capable of distinguishing between the activities of ZAP and other PTKs would be useful especially since it has recently been shown that ZAP interacts with another cell PTK lck We have thus identified site of phosphorylation on the cytoplasmic fragment of the erythrocyte band protein that is recognized by ZAP but not lck synthetic peptide based on this site has been demonstrated to be good in vitro substrate for ZAP and poor substrate for the cell PTKs lck and itk this peptide molecule should thus prove useful to many investigators working in the field of cell activation

A fucoxanthin chlorophyll protein FCP cDNA from the raphidophyte heterosigma carterae encodes amino-acid polypeptide that has similarity to other FCPs and to the chlorophyll binding protein CABs of terrestrial plants and green algae the putative transit sequence has characteristics that resemble signal sequence the heterosigma fcp gene are part of large multigene family which includes members encoding at least two significantly different polypeptides fcp1 fcp2 comparison of the FCP sequence to the recently determined three dimensional structure of the pea LHC II complex indicates that many of the key amino-acid thought to participate in the binding of chlorophyll and the formation of complex stabilizing ionic interactions are well conserved phylogenetic analyses of sequence of lightharvesting protein shows that the FCPs of several chromophyte phyla form natural group separate from the intrinisic peridinin chlorophyll protein iPCPs of the dinoflagellates although the FCP and CAB gene shared common ancestor these lineages diverged from each other prior to the separation of the CAB LHC and LHC II sequence in the green algae and terrestrial plants

We have developed methodologies for the determination of sites of protein phosphorylation by tandem microbore column chromatography with on line detection by electrospray ionization mass spectrometry ESI MS of phosphopeptides recovered from two dimensional 2D phosphopeptide maps We have applied these method to the identification of sites of tyrosine phosphorylation induced on the lymphocyte specific protein tyrosine kinase ZAP and the cell receptor TCR subunit in vivo following stimulation of cell via their TCR our approach is generally applicable to the investigation of signal transduction pathways since it involves direct ESI MS analysis of peptide recovered from 2D phosphopeptide maps currently the analytical technique of choice for the detailed analysis of protein phosphorylation sites and is fully adaptable for the analysis of serine and threonine phosphorylated peptide

translation initiation in eukaryotes is facilitated by the mRNA cap structure m7gpppX where is any nucleotide that binds the multisubunit initiation factor eIF4F through one of its subunits eIF4E eIF4E is phosphoprotein whose phosphorylation state positively correlates with cell growth protein kinase phosphorylates eIF4E in vitro and possibly in vivo using recombinant eIF4E incubated in vitro with purified protein kinase and analyzed by solid phase phosphopeptide sequencing in combination with high performance liquid chromatography coupled to mass spectrometry we demonstrated that the third amino-acid of the peptide SGSTTK ser209 is the major site of phosphorylation this finding is consistent with the newly assigned in vivo phosphorylation site of eIF4E joshi cai keiner minich mendez beach stepinski stolarski darzynkiewicz and rhoads biol chem S209A mutation resulted in dramatically reduced phosphorylation both in vitro and in vivo furthermore the mutant protein was phosphorylated on threonine most probably threonine in vivo here we show that in the presence of the recently characterized translational repressors 4E BP1 or 4E BP2 phosphorylation of eIF4E by protein kinase is strongly reduced this suggests two step model for the phosphorylation and activation of eIF4E by growth factors and hormones first dissociation of eIF4E from 4E BPs followed by eIF4E phosphorylation

We describe an analytical system for the rapid identification of protein by correlation of tandem mass spectra with protein sequence databases the system consists of an integrated solid phase microextraction capillary zone electrophoresis peptide separation device that is connected through microelectrospray ion source to tandem mass spectrometer the limits of detection are amol of sample at concentration limit of amol µl for peptide mass measurement and fmol of sample at concentration limit of amol µl for peptide analysis by collision induced dissociation using this system we have identified low nanogram amounts of yeast protein separated by high resolution two dimensional gel electrophoresis

the terminal src kinase CSK is ubiquitously expressed cytosolic enzyme capable of phosphorylating and inactivating several plasma membrane bound src family protein tyrosine kinases in ritro nada okada macauley cooper and nakagawa nature bergman mustelin oetken partanen flint amrein autero burn and alitalo EMBO We purified CSK to apparent homogeneity from bovine thymus cytosol to study in vitro how the purified enzyme recognizes the various src family kinases as its substrates novel assay method was developed for assaying the ability of CSK to inactivate src family tyrosine kinases with this assay method we demonstrated that CSK inactivated p56 lyn with significantly higher efficiency than pp60 src phosphopeptide mapping of CSK phosphorylated p56 lyn and pp60 src shows that the consensus tyrosine residue also termed tail tyrosine in the terminal regulatory domain of p56 lyn was phosphorylated by CSK with an efficiency much higher than that of pp60 src thus the higher efficiency of inactivation of p56 lyn by CSK is result of the ability of p56 lyn to serve as better substrate of CSK the synthetic peptide derived from the terminal portion of p56 lyn and the pp60 src were much poorer substrates than the intact src family kinases for CSK indicating that the local structure around the tail tyrosine is not sufficient to direct efficient phosphorylation of p56 lyn by CSK nevertheless the slightly higher efficiency displayed by CSK in phosphorylating the peptide derived from the terminal portion of p56 lyn than that from pp60 src suggests that the structural differences between the terminal portions of p56 lyn and pp60 src contribute to the different efficiencies displayed by CSK in phosphorylating the two kinases determination of the CSK phosphorylation site in the src terminal peptide by phosphopeptide mapping reveals that the whole terminal regulatory domain and an adjacent part of the protein kinase domain contain some of the structural determinants directing CSK to phosphorylate the consensus tail tyrosine of the src family kinases

the ZAP protein tyrosine kinase is essential for cell antigen receptor TCR mediated signaling the absence of ZAP results in impaired differentiation of cell and lack of responsiveness to antigenic stimulation In order to study the characteristics of ZAP in vitro we overexpressed an epitopically tagged human ZAP in recombinant baculovirus expression system and purified it by column chromatography the kinase activity of purified recombinant ZAP required cation and exhibited strong preference for Mn2+ over Mg2+ the apparent of ZAP for ATP was µM the activity of the recombinant ZAP unlike that of the homologous protein tyrosine kinase syk was not affected by binding of TCR derived tyrosine phosphorylated immunoreceptor tyrosine based activation motif peptide several protein were tested as potential in vitro substrates of ZAP only tubulin and the cytoplasmic fragment of human erythrocyte band cfb3 which have region of sequence identity at the phosphorylation site proved to be good substrates exhibiting values of and µM respectively ATP µM and casein were poor substrates for ZAP and no activity toward enolase myelin basic protein calmodulin histone protein or angiotensin could be detected In contrast to the cell protein tyrosine kinase lck ZAP did not phosphorylate the cytoplasmic portion of the TCR chain or short peptide corresponding to the CD3e or the TCR immunoreceptor tyrosine based activation motifs our studies suggest that ZAP exhibits high degree of substrate specificity

A detailed structural analysis of the serum amyloid protein SAA of an individual with highly active chronic rheumatoid arthritis is reported SAA isoforms were separated by high resolution two dimensional gel electrophoresis peptide mapping by reverse phase chromatography electrospray ionization tandem mass spectrometry was applied to correlate the protein contained in each spot with their respective coding gene and to study the posttranslational processing and modification events which might result in differential electrophoretic mobility nine protein spots were analyzed the six major spots corresponded to the arg and des arg forms of SAA1a and SAA2a respectively and to the glycosylated and nonglycosylated form of constitutive serum amyloid protein SAA two minor spots were identified as SAA1a isoforms containing post translational modifications We suggest that these variants contained dimethylasparagine residue at position and that one of them was additionally oxidized at trp53 and trp85 the ninth spot was shown to contain mixture of SAA1a and SAA2a To our knowledge this is the first report in which analysis of peptide has been used to verify the presence of SAA in acute phase serum furthermore the data illustrate that extensive post translational processing results in structurally diverse class of acute phase SAA protein which are derived from small number of gene finally the fast and conclusive technology used in this study promises to be generally useful for the comprehensive investigation of protein at the level of the primary structure

A method for the identification of protein by their amino-acid sequence at the low femtomole to subfemtomole sensitivity level is described It is based on an integrated system consisting of capillary zone electrophoresis CZE instrument coupled to an electrospray ionization triple quadrupole tandem mass spectrometer ESI MS MS via microspray interface the method consists of proteolytic fragmentation of protein peptide separation by CZE analysis of separated peptide by ESI MS MS and identification of the protein by correlation of the collision induced dissociation CID patterns of selected peptide with the CID patterns predicted from all the isobaric peptide in sequence database using standard peptide applied to µm capillary we demonstrate an ESI MS limit of detection of less than amol and CID spectra suitable for searching sequence databases obtained with amol of sample applied to the capillary successful protein identification by the method was demonstrated by applying and fmol of tryptic digest of the protein lactoglobulin and bovine serum albumin respectively to the system method for the identification of protein by their amino-acid sequence at the low femtomole to subfemtomole sensitivity level is described It is based on an integrated system consisting of capillary zone electrophoresis CZE instrument coupled to an electrospray ionization triple quadrupole tandem mass spectrometer ESI MS MS via microspray interface the method consists of proteolytic fragmentation of protein peptide separation by CZE analysis of separated peptide by ESI MS MS and identification of the protein by correlation of the collision induced dissociation CID patterns of selected peptide with the CID patterns predicted from all the isobaric peptide in sequence database using standard peptide applied to µm capillary we demonstrate an ESI MS limit of detection of less than amol and CID spectra suitable for searching sequence databases obtained with amol of sample applied to the capillary successful protein identification by the method was demonstrated by applying and fmol of tryptic digest of the protein lactoglobulin and bovine serum albumin respectively to the system

the production survival and function of monocytes and macrophages is regulated by the macrophage colony stimulating factor CSF or CSF through its tyrosine kinase receptor fms binding of CSF to fms induces the tyrosine phosphorylation and association of kD protein with the phosphotyrosine binding PTB domain of she We have cloned p150 using modified yeast two hybrid screen p150 contains one SH2 domain two potential PTB binding sites an ATP GTP binding domain several potential SH3 binding sites and domain with homology to inositol polyphosphate phosphatases p150 antibodies detect this protein in FDC P1 myeloid cell but the same protein is not detectable in fibroblasts the antibodies immunoprecipitate kD protein from quiescent or CSF stimulated FDC P1 cell that hydrolyzes ptdins P3 to ptdins P2 this activity is observed in she immunoprecipitates only after CSF stimulation retroviral expression of p150 in FD fms cell results in strong inhibition of cell growth in CSF and lesser inhibition in IL ectopic expression of p150 in fibroblasts does not inhibit growth this novel protein p150ship SH2 containing inositol phosphatase identifies component of new growth factor receptor signaling pathway in hematopoietic cell

We have identified the residue in cellulomonas fimi exoglycanase modified by bromoacetyl cellobiosylamine as glu using new combination of experimental approaches the enzyme was quantitatively inhibited with the affinity label bromoacetyl cellobiosylamine and cleaved with pepsin the acetyl cellobiosylamine modified peptide was identified by comparative peptide mapping of the digests derived from labeled and unlabeled protein by reverse phase high performance liquid chromatography connected on line to an electrospray ionization mass spectrometer the modified residue in the labeled peptide was determined by using novel protein sequencing chemistry which is based on monitoring the amino-acid derivatives released by stepwise peptide degradation using electrospray ionization mass spectrometry tandem mass spectrometry was used for further structural characterization of the cleaved residue We show that the residue modified by bromoacetyl cellobiosylamine is glu this residue has been identified previously as the acid base catalyst by using combination of mutagenic and kinetic analyses our results therefore demonstrate the usefulness of this type of affinity label in identifying important catalytic residue in glycosidases and suggest that this new experimental approach can be applied generally to any labeled protein in which the mass of the label is known and thus represents an alternative approach to the current method used to identify labeled residue within protein

p56lck is potential in vivo substrate for the tyrosine specific phosphatase CD45 In this study recombinant purified p56lck was found to specifically associate with recombinant CD45 cytoplasmic domain protein but not to the cytoplasmic domain of another related tyrosine phosphatase receptor protein tyrosine phosphatase under equilibrium binding conditions the binding was saturable and occurred at molar stoichiometry fusion protein containing only the amino terminal region of p56lck residue also bound to recombinant CD45 and further analysis of this region indicated that glutathione transferase fusion protein of the unique amino terminal region and the SH2 domain but not the SH3 domain of p562lck bound to recombinant CD45 the SH2 domain protein bound with higher affinity than the amino terminal region but both were able to compete for the binding of p56lck to CD45 and when added together worked synergistically to compete for p56lck binding the SH2 domain interaction with CD45 was specific as glutathione transferase SH2 fusion protein from p85a subunit of phosphatidylinositol kinase and SHC did not bind to CD45 In addition this interaction occurred in the absence of any detectable tyrosine phosphorylation on CD45 suggesting nonconventional SH2 domain interaction

the glucosidase from agrobacterium sp catalyzes the hydrolysis of glucosides via covalent glucopyranosyl enzyme intermediate involving glu358 hydrolysis of dinitrophenyl glucopyranoside by the low activity glu358asp mutant of agrobacterium glucosidase is accompanied by time dependent inactivation of the enzyme through kinetic studies labeling and sequence analysis inactivation is shown to be consequence of the occasional time in attack of tyr298 on the anomeric center of the substrate in place of the catalytic nucleophile with formation of stable glucopyranosyl tyrosine residue tyr298 is conserved throughout family of glycoside hydrolases an indication of possible role in catalysis results of kinetic analysis of the tyr298phe mutant are consistent with function of tyr298 in both orienting the nearby nucleophile glu358 and stabilizing its deprotonated state in the free enzyme

SHP and SH PTP2 are related cytoplasmic protein tyrosine phosphatases having two tandem aminoterminal src homology domains linked to single catalytic domain there is growing evidence that these two molecules may exbibit opposing effects within specific signaling pathways however the relative contributions of the src homology domains or the catalytic domains to these opposing effects are not well known To evaluate the potential contribution of the catalytic domains we compared the substrate specificity of the two phosphatases As seen previously the catalytic activities of bacterially expressed SHP and SH PTP2 were regulated by the presence of the linked src homology domains In addition we characterized cryptic thrombin cleavage site within the carboxy terminus of SHP that led to striking increase in the activity of the catalytic domain employing panel of phosphopeptide substrates whose sequence were modeled after intracellular phosphorylation sites both SHP and SH PTP2 demonstrated similar specificity pattern similar to SH PTP2 SHP failed to elicit detectable phosphate release from several phosphopeptide substrates while displaying catalytic efficiencies that ranged over towards other substrates In contrast the PTP 1B phosphatase dephosphorylated all of the phosphopeptide substrates tested with approximately equal ease the overall similarity demonstrated by the catalytic domains of SHP and SH PTP2 suggested that differences in the in vivo behavior of these two molecules might not stem from differences in the substrate specificity of the catalytic domains suggesting instead that the specificity of the src homology domains is more important in this regard

A new method that uses nonradioactive active site directed enzyme inactivators and high performance liquid chromatography electrospray ionization tandem mass spectrometry HPLC ESIMS MS to identify labeled peptide in proteolytic digest is described this method relies upon the fragmentation of labeled peptide in predictable and reproducible manner in the collision cell of tandem mass spectrometer the exoglycanase from cellulomonas fimi endoglucanase from clostridium thermocellum and the glucosidase from agrobacterium faecalis were labeled using deoxy halo glycosides digested with pepsin and subjected to HPLC ESIMS MS analysis scanning in the neutral loss mode under these conditions only peptide that lose the known mass of the label are detected preliminary identification of candidate peptide can be achieved from the mass measured in combination with the known sequence of the protein peptide identity can be confirmed through subsequent sequencing either via further tandem MS experiments or via the edman degradation In all cases the peptide identified in this manner were consistent with those identified by the standard radioactive method this mass spectrometric method represents rapid nonradioisotopic solution to the problem of identifying modified peptide in complex mixture the technique is also sensitive requiring only picomole amounts of protein

We have previously reported that human colon cancer cell secrete factor which induces elongation of colon fibroblasts in vitro isolation of this factor led to the identification of 55kD protein with fibroblast stretching activity two internal amino-acid sequence identified in this protein YEI GFQALGDAADI share complete homology with tissue inhibitor of metalloproteinase TIMP

We report the synthesis and structural characterization of the novel edman type protein sequencing reagent pyridinylmethylaminocarboxypropyl phenyl isothiocyanate panel of thiohydantoins prepared from this reagent were found stable during liquid chromatography electrospray mass spectrometry and were detectable at the low femtomole sensitivity level furthermore the signal detected for these compounds in the mass spectrometer was linear from the low femtomole to the low picomole range the derivatives showed uv absorbance spectra comparable to their phenylthiohydantoin counterparts the extinction coefficient for the pyridinylmethylaminocarboxypropyl phenyl thiohydantoin tyrosine was determined by adsorptive sequence analysis of synthetic pentapeptide featuring an terminal 125I labeled tyrosine the sequence data suggest that the reagent will be useful for extended sequence analysis of protein and peptide using commercially available gas liquid phase sequencers

We report the separation of pyridinylmethylaminocarboxypropyl phenylthiohydantoins by microbore reverse phase high performance liquid chromatography and their detection by on line electrospray ionization mass spectrometry these compounds are the products of the chemical stepwise degradation of polypeptides using pyridinylmethylaminocarboxypropyl phenyl isothiocyanate We describe chromatographic conditions for on column concentration of the analytes and for baseline separation of the isobaric amino-acid derivatives of leucine and isoleucine commercially available protein sequencer was readily interfaced with the described analytical system and used for adsorptive sequence analysis of panel of synthetic peptide containing collectively all naturally occurring amino-acid On line mass analysis of derivatives generated by automated sequencing confirmed that the derivatives were of the predicted mass and were detectable at comparable signal strength and sensitivity finally we demonstrate that the additional selectivity in data interpretation provided by mass analysis dramatically improves the signal to noise ratio and therefore enhances the ability to conclusively interpret protein and peptide sequence data

signaling by the cell antigen receptor TCR is mediated by residue tyrosine based activation motifs TAM present in the cytoplasmic tails of the TCR and CD3 chains TAMs become tyrosine phosphorylated upon TCR stimulation creating high affinity binding site for the tandem SH2 domains of ZAP In permeabilized cell the association of TCR and ZAP was inhibited by protein tyrosine phosphatase PTpase resistant TAM peptide analog in which difluorophosphonomethyl phenylalanyl F2pmp residue replaced phosphotyrosine inhibition of this association prevented TCR stimulated tyrosine phosphorylation of ZAP and reduced ZAP kinase activity to basal levels the reduction in ZAP activity coincided with reduced tyrosine phosphorylation of number of substrates such PTpase resistant peptide capable of disrupting SH2 domain mediated protein protein interactions should prove useful in further dissection of multiple signaling pathways and may serve as models for rationally designed chemotherapeutic agents for the treatment of autoimmune and neoplastic disorders

the insulin regulated glucose transporter isotype gluT4 expressed only in muscle and adipose cell is sequestered in specific secretory vesicle these vesicles harbor another major protein referred to as vp165 for vesicle protein of kDa that like gluT4 redistributes to the plasma membrane in response to insulin We describe here the cloning of vp165 and show that it is novel member of the family of zinc dependent membrane aminopeptidases with the typical large extracellular catalytic domain and single transmembrane domain but with unique extended cytoplasmic domain the latter contains two dileucine motifs which may be critical for the specific trafficking of vp165 since this has been shown to be the case for this motif in gluT4 however the tissue distribution of vp165 is much wider than that of gluT4 consequently vp165 may also function in process unrelated to insulin action and may serve as ubiquitous marker for specialized regulated secretory vesicle

engagement of the cell antigen receptor TCR results in activation of several tyrosine kinases leading to tyrosine phosphorylation of protein substrates and activation of multiple biochemical pathways TCR mediated activation of the src family kinases lck and fyn results in tyrosine phosphorylation of the TCR and CD3 chains the site of phosphorylation in these chains is the tyrosine based activation motif TAM amino-acid module containing two tyrosine residue tyrosine phosphorylated TAMs serve as targets for binding of the zeta associated protein ZAP tyrosine kinase via its tandem SH2 domains this binding correlates with activation of ZAP critical event in cell activation To further define the structural requirements for ZAP interaction with the TCR we developed binding assay using immobilized glutathione transferase fusion protein containing the NH2 and or COOH terminal SH2 domains of ZAP and soluble synthetic peptide with the sequence of the cytoplasmic region of the TCR chain TCR cyt or individual TCR and CD3e TAM motifs direct binding studies demonstrated that the tandem ZAP SH2 domains bind phosphorylated but not nonphosphorylated TCR cyt the NH2 terminal ZAP SH2 domain also binds to TCR cyt but with fold lower affinity No binding was observed with the COOH terminal ZAP SH2 domain similar studies demonstrated that the ZAP tandem SH2 domain can bind TCR TAM peptide in which both tyrosine residue are phosphorylated little or no binding was observed with peptide phosphorylated at only one tyrosine residue or nonphosphorylated peptide binding of the tandem SH2 domains to the other two TCR TAM peptide and to CD3e TAM peptide was also observed all four doubly tyrosine phosphorylated TAM peptide cross compete with each other for binding to the tandem SH2 domains of ZAP the affinity of these peptide for the tandem SH2 construct demonstrated hierarchy of TAM TAM gt TAMe TAM the results provide further evidence that the ZAP interaction with the TCR requires prior phosphorylation of both tyrosine residue within TAM motif binding of ZAP to phospho TAMs is notable for the high level of cooperativity between the two SH2 domains which individually demonstrate low affinity interaction with the ligand the cooperativity ensures higher affinity for the doubly phosphorylated ligand affinity differences of as much as fold indicates significant specificity of interaction of ZAP SH2 domains for different phospho TAMs

glycosidases play key role in number of biological process and as such are of considerable clinical and biotechnological importance knowledge of the identities of catalytically important active site residue is essential for understanding the catalytic mechanism for enzyme classification and for targeted bioengineering of glycosidases with altered characteristics here we review and discuss traditional strategies and novel approaches based on tandem mass spectrometry for the identification of the key active site residue in glycosidases

We describe simple fast sensitive and nonisotopic bioanalytical technique for the detection of tyrosine phosphorylated peptide and the determination of sites of protein tyrosine phosphorylation the technique employs protein tyrosine phosphatase micro enzyme reactor coupled on line to either capillary electrophoresis or liquid chromatography and electrospray ionization mass spectrometry instruments the micro enzyme reactor was constructed by immobilizing genetically engineered metabolically biotinylated human protein tyrosine phosphatase onto the inner surface of small piece of µm inner diameter µm outer diameter fused silica capillary or by immobilization of the phosphatase onto µm avidin activated resins By coupling these reactors directly to either capillary electrophoresis column or liquid chromatography column we were able to rapidly perform enzymatic dephosphorylation and separation of the reaction products detection and identification of the components of the reaction mixture exiting these reactors were done by mass analysis with an on line electrospray ionization mass spectrometer tyrosine phosphorylated peptide even if present in complex peptide mixture were identified by subtractive analysis of peptide patterns generated with or without phosphatase treatment two criteria namely phosphatase induced change in hydropathy and charge respectively and change in molecular mass by Da were used jointly to identify phosphopeptides We demonstrate that with this technique low picomole amounts of tyrosine phosphorylated peptide can be detected in complex peptide mixture generated by proteolysis of protein and that even higher sensitivities can be realized if more sensitive detection system are applied

ZAP is kDa protein tyrosine kinase expressed exclusively in cell and NK cell and plays critical role in mediating cell activation in response to cell receptor engagement the strong correlation between tyrosine phosphorylation of ZAP and its acquisition of increased kinase activity suggests that it is positively regulated by tyrosine phosphorylation previously we identified tyrosines and of ZAP as being sites of in vivo phosphorylation in response to cell receptor engagement To determine the role of phosphorylation in regulating ZAP activity we mutated each of these tyrosines individually to phenylalanine when expressed in COS cell Y493F mutated ZAP demonstrated normal basal kinase activity but unlike wild type ZAP could not be activated by tyrosine phosphorylation induced by incubation with pervanadate or by co expression of constitutively activated lck this suggests that tyr phosphorylation is required for the tyrosine phosphorylation induced activation of ZAP the Y492F mutation resulted in fold higher basal kinase activity which could he stimulated further by tyrosine phosphorylation these results reveal that critical tyrosine residue in the kinase domain of ZAP are important in regulation of its catalytic activity

A new mechanism based inactivator of xylanases dinitrophenyl deoxy fluoro xylobioside has been synthesized and used to trap the covalent intermediate formed during catalysis by bacillus subtilis xylanase electrospray mass spectrometry confirmed the stoichiometry of the incorporation of inactivator into the enzyme inactivation of xylanase followed the expected pseudo first order kinetic behavior and kinetic parameters were determined the intermediate trapped was relatively stable toward hydrolytic turnover t1 min however turnover could be facilitated by transglycosylation following the addition of the acceptor benzyl thio xylobioside thus demonstrating the catalytic competence of the trapped intermediate reactivation kinetic parameters for this process of kre min and kre mM were determined the nucleophilic amino-acid was identified as glu78 by tandem mass spectrometric technique which does not require the use of radiolabels the peptic digest of the labeled enzyme was separated by high performance liquid chromatography and the eluent fed into tandem mass spectrometer via an electrospray ionization device the labeled peptide was identified as one of doubly charged which fragmented in the collision chamber between the mass analyzers with loss of the mass of fluoroxylobiosyl unit confirmation of the peptide identity was obtained both by tandem mass spectrometric sequencing and by edman degradation of the purified peptide glu78 is completely conserved in all members of this xylanase family and indeed is shown to be located in the active site in the recently determined ray crystal structure

We have developed rapid and sensitive two capillary column chromatography and mass spectrometry based method for the determination of protein phosphorylation sites following recovery of individual phosphopeptides from two dimensional phosphopeptide maps with standard phosphopeptide we demonstrate detection sensitivity of at least fmol for this system We applied this technique to the analysis of in vitro sites of tyrosine phosphorylation induced on the cell specific protein tyrosine kinase ZAP in the absence and presence of p56 lck We show that ZAP has primary autophosphorylation site at tyr with secondary site at tyr We also show additional phosphorylation at tyr tyr tyr and tyr upon the addition of the protein tyrosine kinase p56 lck By comparative two dimensional phosphopeptide mapping we show that ZAP isolated from jurkat cell also autophosphorylates at tyr and tyr similar analysis of 32P labeled jurkat cell stimulated with anti cell receptor antibodies reveals tyr and tyr as the principal sites of cell antigen receptor induced tyrosine phosphorylation with additional phosphorylation at the tyr but not the tyr autophosphorylation site the high degree of sensitivity achieved with this technology should greatly facilitate the direct biochemical determination of inducible protein phosphorylation events an experimental strategy that until now has been both time consuming and difficult

phosphorylation of the neurofilament protein of high and medium relative molecular mass as well as of the alzheimer tau protein is thought to be catalysed by protein kinase with cdc2 like substrate specificity We have purified novel cdc2 like kinase from bovine brain capable of phosphorylating both the neurofilament protein and tau the purified enzyme is heterodimer of cyclin dependent kinase cdk5 and novel regulatory subunit p25 when overexpressed and purified from escherichia coli p25 can activate cdk5 in vitro unlike cdk5 which is ubiquitously expressed in human tissue the p25 transcript is expressed only in brain full length complementary DNA clone showed that p25 is truncated form of larger protein precursor p35 which seems to be the predominant form of the protein in crude brain extract cdk5 p35 is the first example of cdc2 like kinase with neuronal function

We show that cross linking the cell AgR with anti Ig abs activates p56lck lck in both the immature cell line WEHI and mature resting cell from mouse spleen anti Ig stimulated lck activity peaked after to min but remained elevated for at least min consistent with the proposed role for src family tyrosine kinases in AgR signaling we found that lck could phosphorylate the cytoplasmic tails of the Ig and Ig components of the cell AgR in vitro lck phosphorylated both of the tyrosines in the Ig AgR homology motif and one of the two tyrosines in the Ig AgR homology motif finally we show that AgR ligation in cell caused significant portion of the lck to migrate with an apparent molecular mass of kDa on SDS PAGE gels conversion of p56lck to p60lck was maximal at to min at which times lck activity in the cell was decreasing this lck band shift has been observed previously in activated cell and correlates with phosphorylation of lck at serine We show that the kDa form of lck induced by AgR cross linking in cell is also phosphorylated at serine phosphorylation of lck at this site in vitro decreases its activity thus in cell AgR cross linking activates lck and subsequently activates kinase that phosphorylates lck at serine potential negative regulatory site

In this report we describe procedure for the isolation of peptide fragments derived by proteolytic cleavage of small amounts of protein separated by polyacrylamide gel electrophoresis protein resolved by gel electrophoresis were isolated by electroblotting onto carrier matrix and digested on the support the released peptide were separated by reverse phase high performance liquid chromatography and collected chromatography fractions were analyzed with respect to peptide homogeneity by capillary electrophoresis or electrospray mass spectrometry and if necessary further separated by rechromatography selected examples illustrate that individual steps of the procedure are mutually compatible and unnecessary sample manipulation can therefore be avoided the described procedure is sensitive simple and generally applicable and is ideally suited to use in the preparation of peptide samples for internal sequencing peptide isolated by these protocols are equally suitable for solving other analytical problems in protein biochemistry such as the determination and localization of modified amino-acid residue and the determination of catalytic residue in active sites of enzymes finally we demonstrate that detailed analysis of peptide maps can complement and in some cases replace protein sequencing

ligation of the cell AgR activates p21ras ras We have investigated the effects of AgR ligation on three protein that have been implicated as regulators of ras SHC GRB and mSOS1 We show that AgR cross linking in cell stimulated tyrosine and serine phosphorylation of SHC this correlated with the formation of complexes containing SHC GRB mSOS1 and an unidentified kDa tyrosine phosphorylated protein these complexes were present in the cytosol as well as in the membrane fraction of the cell where ras is located By using GRB fusion protein to probe blots we showed that SHC was the major protein that GRB bound to in anti Ig stimulated cell this argues that SHC couples GRB mSOS1 to the kDa protein and that SHC is likely to be essential for mSOS1 function in cell finally we found that AgR cross linking stimulated phosphorylation of mSOS1 and that this could be blocked by an inhibitor of protein kinase thus signaling by the cell AgR stimulates phosphorylation of SHC and mSOS1 and induces the formation of membrane associated complexes containing SHC GRB mSOS1 and kDa protein these events may be important for activation of ras by the AgR

gaucher disease is an inherited lysosomal storage disorder caused by deficiency of human acid glucosidase glucocerebrosidase this enzyme is inactivated by the specific mechanism based enzyme inactivator deoxy fluoro glucopyranosyl fluoride which functions by forming stable deoxy fluoro glucopyranosyl enzyme intermediate the key nucleophilic amino-acid residue involved in formation of this intermediate was conclusively identified by tandem mass spectrometry as glu340 and not asp443 as thought previously this was confirmed by site directed mutagenesis identification and mass determination of the labeled peptide in proteolytic hydrolysate involved detection of collision induced fragmentation reaction specific to the sugar peptide linkage confirmation of the identity of the labeled peptide was obtained both by tandem mass spectrometric sequencing and by chemical degradation of the purified peptide this method allowed the rapid sensitive and non isotopic determination of an essential amino-acid residue in clinically important enzyme

transcription factor IIIC TFIIIC is multisubunit basic TF for RNA polymerase III It initiates transcription complex assembly on tRNA and related gene by binding to the internal box promoter element and is also required for transcription of 5S rRNA and other stable nuclear and cytoplasmic RNAs transcribed by polymerase III In mammalian cell regulation of TFIIIC activity controls overall polymerase III transcription in response to growth factors and viral infection here we report the cloning and sequencing of full length cDNA and genomic DNA from the transcription initiation region encoding the box binding subunit of human TFIIIC the kDa subunit specific antisera raised against the encoded protein super shifts TFIIIC box DNA complex during an electrophoretic mobility shift assay and immunodepletes TFIIIC transcriptional activity from partially purified TFIIIC fraction proving that the cDNA encodes component of TFIIIC the human protein shows surprisingly little similarity to the box binding subunit of yeast TFIIIC

GLUT4 enriched vesicles from fat and muscle cell contain major protein of kDa In the present study we have prepared GLUT4 vesicles from large number of rat adipocytes isolated the kDa protein by SDS gel electrophoresis and obtained the sequence of four tryptic peptide comparison of the sequence with those in protein data banks showed that the kDa protein had not previously been sequenced An antibody preparation against one of the peptide immunoblotted the kDa protein By this means it was found that the kDa protein was concentrated in GLUT4 vesicles and that insulin treatment of rat adipocytes caused translocation of the protein to the plasma membrane that paralleled the translocation of GLUT4

T cell receptor TCR chain TCRa and chain TCRß gene are well characterized in mammals while only TCRß gene have been identified in other vertebrates To identify avian TCRa gene we used monoclonal anti CD3 antibodies to isolate chicken TCRa for peptide sequence analysis degenerate oligonucleotide probes were then used to isolate candidate TCRa cDNA clone that hybridized with kb mRNA species present only in aß cell and in tissues populated by these cell southern blot analysis revealed gene rearrangement in thymocytes and aß cell lines the TCRa cDNA candidate encoded an open reading frame of amino-acid the predicted variable joining and constant region amino-acid sequence of which shared and homology with corresponding mammalian sequence single gene and gene were identified by using region specific probes the cDNA probe isolated from 1+ cell line reacted with transcripts from one of five 2+ cell lines suggesting shared use of gene by 1+ and 2+ cell and the existence of other gene families genomic sequence was flanked by classical recombination signal sequence but unlike previously defined gene the leader and region were encoded by single exon the data indicate evolutionary conservation of the basic TCRa gene structure in birds and mammals

A technique is described for the rapid sensitive analysis of posttranslational modifications of protein that have been separated by dimensional electrophoresis and blotted onto membrane with cationic surface the isolated protein spots visualized by reverse staining of the blotting membrane are excised washed and subjected to chemical cyanogen bromide and or enzymatic endoproteinase lys degradation directly on the membrane the resulting mixture of peptide fragments is extracted from the membrane into solution that is compatible with matrix assisted laser desorption mass spectrometric analysis and analyzed without fractionation relatively accurate Da mass determination of these peptide fragments provides facile and sensitive means for detecting the presence of modifications and for correlating such modifications with the differential mobility of different isoforms of given protein during dimensional electrophoresis the technique is applied to the determination of sites of phosphorylation in synapsins Ia and Ib neuronal phosphoproteins that are believed to function in the regulation of neurotransmitter release and are substrates for cAMP and Ca2+ calmodulin dependent protein kinases which appear to control their biological activity

the activation of protein tyrosine kinases PTKs and subsequent tyrosine phosphorylation of cellular protein is critical initial signal in the response of eukaryotic cell to mitogens differentiative signals and other stimuli number of PTK substrates have been identified and many of these are components of signal transduction pathways that regulate cell function however the majority of protein that are tyrosine phosphorylated in response to receptor signaling remain unidentified As some of these unidentified PTK substrates may also be signal transducing protein their identification and functional characterization is an important objective towards understanding receptor signaling We describe the development of comprehensive and general process for the isolation and structural characterization of tyrosine phosphorylated protein the method involves enrichment by anti phosphotyrosine affinity chromatography electrophoretic concentration and separation and proteolytic fragmentation of individual purified phosphoproteins resulting peptide fragments are separated by microbore reverse phase high performance liquid chromatography RP HPLC and portion of the eluted peptide are subjected to electrospray mass spectrometry ES MS for accurate determination of peptide masses proteolytic fragmentation of protein produces characteristic set of peptide masses that can be used to rapidly identify the protein by searching databases containing the peptide mass fingerprints for all known protein the identity of the protein established by this method can be confirmed by sequence analysis of selected peptide We have applied this procedure to the analysis of PTK substrates from lymphocytes that have been stimulated through the cell antigen receptor BCR signaling by this receptor is involved in the generation of antibodies against foreign molecules antigens the BCR activates multiple PTKs which phosphorylate at least different protein We have identified several of these tyrosine phosphorylated protein including syk PTK that is known to be tyrosine phosphorylated in activated cell thus the procedure described here can be used to identify regulatory protein of low abundance the process consists of logical succession of compatible steps that avoids pitfalls inherent to prior attempts to characterize low abundance phosphoproteins and should find wide use for the identification of tyrosine phosphorylated protein in other cell types

aA and aB crystallins are significant contributors to maintaining the transparency of the vertebrate lens We have found that both aA and aB crystallins are also present at approximately equimolar concentrations in frog retinal cell they were identified by sequencing portions of each polypeptide by immunochemical cross reactivity with antibodies to bovine crystallins and by their relative mobility in two dimensional gel electrophoresis retinal crystallins form macromolecular multimeric complexes similar to those found in the lens and they are abundant both in soluble and membrane associated forms surprising finding is that crystallins bind specifically to the photoreceptor post golgi membranes that mediate transport of newly synthesized rhodopsin upon treatment of post golgi membranes with urea or triton portion of the bound aB crystallin remains tightly associated indicating that the aB form may mediate membrane binding of an crystallin multimeric complex both subunits are synthesized in vitro by isolated frog retinas but aB crystallin appears to have higher renewal rate newly synthesized crystallins become associated with the post golgi membranes concurrently with newly synthesized rhodopsin association of crystallins with newly synthesized rhodopsin suggests that they may participate in photoreceptor outer segment membrane renewal our findings implicate an important function for both aA and aB crystallins in the same extralenticular tissue

activation of phosphatidylinositol PI kinase is common sequel to tyrosine kinase activation and appears to be essential for tyrosine kinases to induce proliferation since multiple hemopoietic growth factors activate tyrosine kinases we investigated whether these growth factors activate PI kinase We show that interleukin IL interleukin IL interleukin IL granulocyte macrophage colony stimulating factor GM CSF and steel factor SLF all activate PI kinase these cytokines increased the amount of PI kinase activity that could be immunoprecipitated with anti phosphotyrosine antibodies from the MC mast cell line or from the hemopoietic progenitor cell line FDC P1 increases in this assay frequently correlate with PI kinase activation in vivo To determine directly whether these factors activate PI kinase in vivo we measured the levels of phosphorylated inositol phospholipids in intact 32P labeled MC cell IL IL IL GM CSF and SLF all caused increased synthesis of the PI kinase products phosphatidylinositol bisphosphate and phosphatidylinositol trisphosphate with relative potency of SLF gt gt IL gt IL GM CSF gt IL In contrast IL caused the largest increase in the in vitro anti phosphotyrosine immune complex PI kinase assay thus the in vitro assay does not accurately reflect in vivo activation of PI kinase cytokine treatment did not stimulate tyrosine phosphorylation of either the kDa regulatory subunit or the kDa catalytic subunit of PI kinase and is therefore not required for activation of PI kinase by these factors cytokine treatment did induce PI kinase to associate with other tyrosine phosphorylated protein in cytokine specific manner PI kinase associated with kit after SLF stimulation kDa protein after IL stimulation and kDa protein after treatment with IL or GM CSF thus multiple hemopoietic growth factors that act through different types of receptors activate PI kinase in vivo and induce factor specific interactions of PI kinase with other tyrosine phosphorylated protein

intracellular signaling pathways are to large extent regulated by reversible protein phosphorylation of pathway components To fully investigate the regulation of these pathways it is often necessary to identify the sites of protein phosphorylation induced on individual components the low abundance of many of these molecules and the potentially low stoichiometry of phosphorylation means that conventional analytical techniques are incapable of identifying specific sites of modification inducible in vivo the most common technique used is two dimensional 2D phosphopeptide mapping electrophoresis thin layer chromatography of peptide derived by proteolysis of phosphoprotein the number of spots detected is commonly interpreted as the number of sites of phosphorylation here we have achieved positive identification of phosphorylation sites by capillary high performance liquid chromatography with on line mass spectrometric detection of phosphopeptides recovered from 2D phosphopeptide maps We demonstrate that the chemical composition of phosphopeptides is not altered during the 2D mapping procedure By detailed analysis of the sites of phosphorylation induced in vitro on CD3 by p56 lck we demonstrate that interpretation of the sites of phosphorylation based on 2D phosphopeptide mapping alone is difficult To minimize over or misinterpretation of 2D phosphopeptide maps we therefore postulate rules that should be applied generally in cases in which protein phosphorylation sites are being evaluated by 2D phosphopeptide patterns alone

the intracellular domain of human protein tyrosine phosphatase HPTPß kDa was expressed in bacteria purified using epitope tagging immunoaffinity chromatography and characterized with respect to kinetic profile substrate specificity and potential modulators of enzyme activity chromogenic assay based on the malachite green method was employed for the detection of inorganic phosphate released from phosphopeptides by HPTPß this assay modified so as to improve its sensitivity was adapted to well microtitre plate format and provided linear detection between and pmol of the cytoplasmic domain of HPTPß was strongly inhibited by vanadate molybdate heparin poly glu tyr and zinc ions In order to explore the substrate preferences of this PTpase we generated residue synthetic phosphotyrosine containing peptide that corresponded to sites of physiological tyrosine phosphorylation HPTPß demonstrated cat values between and using four different phosphopeptides the substrate preference of HPTPß was in the order src tyr gt PDGF tyr gt ERK1 tyr gt gt CSF 1R tyr with values ranging from µM to greater than mM the variations in affinity were probably due to differences among the four phosphopeptides compared particularly with respect to the character of the charged amino-acid flanking the phosphotyrosine residue

rat liver acetyl CoA carboxylase ACC EC exhibits major and minor subunits of and respectively the structure and function of which are compared in this study the two subunits copurified and each contained biotin as demonstrated by avidin reactivity and direct determination of biocytin In agreement with previous studies the ACC subunits could be distinguished with specific monoclonal antibodies and differential tissue expression We now report extensive differences in primary structure revealed by peptide mapping mass spectrometric analysis of peptide following reverse phase high performance liquid chromatography and microsequencing of selected peptide four peptide derived from the kDa subunit were sequenced and matched sequence within the predicted structure of rat kDa ACC although one identical peptide sequence was detected within both subunits residue of the kDa subunit peptide derived from the kDa subunit exhibited entirely novel sequence or matched partially average identity with sequence within the kDa subunit the kDa subunit may also exhibit distinct functional properties since the initial rate of phosphorylation was at least fold greater than that of the kDa subunit in the presence of cAMP dependent protein kinase two dimensional mapping demonstrated that the tryptic phosphopeptides released from the two ACC subunits are distinct these structural studies suggest that the and kDa components isozymes of ACC are so distinct they may be encoded by separate gene while the differential phosphorylation observed in vitro suggests key role for the kDa subunit in regulating enzyme activity within intact cell

the effects of Ag binding on cell development and activation are mediated by intracellular signals initiated by the cell AgR In this report we show that the cell AgR regulates the production of inositol phospholipids involved in two different signal transduction pathways the phosphatidylinositol kinase ptdins kinase pathway and the phospholipase PLC pathway phosphatidylinositol phosphate ptdins3P phosphatidylinositol bisphosphate ptdins and phosphatidylinositol trisphosphate ptdins are produced by ptdins kinase an enzyme that appears to be an essential component of tyrosine kinase mediated signaling both ptdins and ptdins are likely to function as second messengers in vivo because they can activate the isoform of protein kinase PKC in vitro We show that cross linking of the cell AgR with anti Ig antibodies caused five to sixfold increase in the levels of ptdins in both the mature cell line BAL and the immature cell line WEHI ptdins levels increased within of anti Ig addition and remained elevated for at least min AgR cross linking also caused slower increase in ptdins3P levels approximately over control and small transient increase in ptdins levels thus the cell AgR activates the ptdins kinase pathway the other inositol phospholipid signaling pathway involves PLC which cleaves phosphatidylinositol bisphosphate ptdins yielding second messengers that increase intracellular calcium and activate other isoforms of PKC We analyzed the effects of AgR signaling on ptdins and its precursor phosphatidylinositol phosphate ptdins4P consistent with its ability to activate PLC AgR ligation decreased the levels of ptdins In contrast AgR cross linking increased the levels of ptdins4P increased synthesis of ptdins4P followed by phosphorylation at the position may prevent depletion of ptdins thus signaling by the cell AgR increases the levels of ptdins kinase products and ptdins kinase products the simplest interpretation of our results is that the cell AgR activates both ptdins kinase and ptdins kinase

p56lck member of the src family of non receptor protein tyrosine kinases is expressed almost exclusively in cell of lymphoid origin p56lck is known to associate with the lymphocyte surface glycoproteins CD4 and CD8 and plays critical role in both lymphocyte development and activation p56lck also associates with the subunit of the IL 2R and is activated when IL binds to its receptor using primary cultures of con activated normal splenic mouse lymphocytes we observed an IL induced sequence of events involving p56lck We saw rapid within to min and transient increase in p56lck kinase activity which preceded the activation of both the p42erk and p44erk mitogen activated protein kinases maximal activation of which was observed after min We also observed an IL induced shift in the electrophoretic mobility of p56lck from an apparent molecular mass of kDa p56lck to kDa p60lck which reached maximum at min the level of p60lck remaining constant for up to thereafter this IL induced shift correlated with the phosphorylation of serine of p56lck site that mitogen activated protein kinases are capable of modifying in vitro the implications of these results for the understanding of both p56lck function and lymphoid cell receptor signaling pathways are discussed

p56lck member of the src family of non receptor protein tyrosine kinases is expressed almost exclusively in cell of lymphoid origin recent evidence has implicated p56lck in critical role both in cell development and activation variety of cell stimuli induce shift in the electrophoretic mobility of p56lck from an apparent molecular mass of kDa p56lck to kDa p60lck this shift in electrophoretic mobility correlates with an increase in the phosphoserine content of the p60lck We have shown that both 4a phorbol 12ß myristate acetate and OKT3 treatment of jurkat cell as well as 4a phorbol 12ß myristate acetate treatment of CD4 and LSTRA cell induced phosphorylation of serine on p56lck in vivo which correlated with the shift to p60lck We also demonstrated that the same serine residue could be phosphorylated in vitro with mitogen activated protein kinases and that this event was capable of reducing p56lck activity in vitro combined these data suggest novel pathway for the in vivo regulation of p56lck activity

the cell receptor TCR subunit is an important component of the TCR complex involved in signal transduction events following TCR engagement In this study we showed that the TCR chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node cell approximately of the tyrosine phosphorylated TCR phospho precipitated from total cell lysates appeared to be surface associated furthermore constitutive phosphorylation of TCR in cell occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression In lymph node cell that constitutively express tyrosine phosphorylated TCR there was direct correlation between surface TCR associated protein tyrosine kinase PTK activity and expression of phospho TCR stimulation of these cell resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and corresponding increase in the levels of phospho TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes including kDa tyrosine phosphorylated protein the presence of TCR associated PTK activity also correlated with the binding of kDa protein which became tyrosine phosphorylated in vitro kinase assay to tyrosine phosphorylated TCR the cytoplasmic region of the TCR chain was synthesized tyrosine phosphorylated and conjugated to sepharose beads only tyrosine phosphorylated not nonphosphorylated TCR beads were capable of immunoprecipitating the kDa protein from total cell lysates this kDa protein is likely the murine equivalent of human PTK ZAP which has been shown to associate specifically with phospho these results suggest that TCR associated PTK activity is regulated at least in part by the tyrosine phosphorylation status of TCR

A new mechanism based inactivator of glucanases dinitrophenyl deoxy fluoro cellobioside was synthesized and used to trap the intermediate formed during catalysis by endoglucanase celC from clostridium thermocellum ion spray mass spectrometry confirmed the stoichiometry of the incorporation of the inactivator into the enzyme inactivation followed the required pseudo first order kinetic behavior and kinetic parameters for the process were determined although the intermediate trapped was relatively stable t1 turnover was facilitated by transglycosylation following the addition of phenyl thiocellobioside and cellobiose thus demonstrating the catalytic competence of the trapped intermediate the nucleophilic amino-acid residue involved was identified as glu280 by labeling the enzyme with tritiated inactivator cleaving it into peptide and sequencing the radiolabeled peptide ion spray mass spectrometric analysis of the peptide confirmed the sequence and the mode of attachment of the sugar to the peptide alignment of all known related glucanases showed that glu280 is strictly conserved in family enzymes consistent with its key role in catalysis

insulin elicits the tyrosine phosphorylation of one or more protein of kDa in many cell types peptide sequence obtained from this protein purified from mouse 3T3 L1 adipocytes pp160 were used as the basis for cloning its cDNA pp160 is highly homologous to the insulin receptor substrate previously cloned from rat liver thus this component of the insulin signaling pathway is the same in adipocytes and in liver

jacalin an IgA binding lectin from jackfruit artocarpus heterophyllus seeds was isolated by the passage of PBS extracts of seeds over an affinity matrix containing IgA sepharose 4B It was further purified by HPLC when analyzed by SDS PAGE under both reducing and nonreducing conditions the native jacalin was dissociated into two subunits of and kDa both the subunits could bind IgA peptide mapping performed with radioiodinated jacalin indicated that both the subunits were susceptible to proteolysis by staphyloccous aureus V8 proteinase one degradation product was small peptide of kDa this small proteolytic fragment also bound IgA the amino termini of the two major IgA binding subunits and kDa were identical the kDa IgA binding proteolytic fragment of jacalin had different amino terminal sequence suggesting that the region of jacalin which binds IgA does not remain close to the amino terminus of the peptide

the gene encoding the subunit of DNA polymerase III holoenzyme holD was identified and isolated by an approach in which peptide sequence data were used to obtain DNA hybridization probe the gene which maps to centisomes was sequenced and found to be identical to previously uncharacterized open reading frame that overlaps the end of rimI by bases contains bp and is predicted to encode protein of Da when expressed in plasmid that also expressed holC holD directed expression of the subunit to about of total soluble protein

the gene encoding the subunit of DNA polymerase III holoenzyme designated holE was isolated using strategy in which peptide sequence was used to derive DNA hybridization probe sequencing of the gene which maps to centisomes of the chromosome revealed codon open reading frame predicted to produce protein of Da when placed in tac promoter expression vector the open reading frame directed expression of protein that comigrated with authentic subunit from purified holoenzyme to of total soluble protein

We report the use of microbore reverse phase high performance liquid chromatography connected on line to an electrospray mass spectrometer for the separation detection of peptide derived by proteolytic digestion of protein separated by polyacrylamide gel electrophoresis small fraction typically of the total of the peptide eluting from the column was diverted through flow splitting device into the ion source of the mass spectrometer whereas the majority of the peptide samples was collected for further analyses We demonstrate the feasibility of obtaining reproducible peptide maps from submicrogram amounts of protein applied to the gel and good correlation of the signal detected by the mass spectrometer with peptide detection by UV absorbance furthermore independently verifiable peptide masses were determined from subpicomole amounts of peptide directed into the mass spectrometer the method was used to analyze the kDa and the kDa isoforms of the enzyme acetyl CoA carboxylase isolated from rat liver the results provide compelling evidence that the two enzyme isoforms are translation products of different gene and suggest that these approaches may be of general utility in the definitive comparison of protein isoforms We furthermore illustrate that knowledge of peptide masses as determined by this technique provides major advantage for error free data interpretation in chemical high sensitivity peptide sequence analysis

A prominent human cerebrospinal fluid CSF protein P5 identified at mass kDa and charge by two dimensional electrophoresis 2DE and silver staining has been previously demonstrated to be reduced in quantity in the CSF of patients with multiple sclerosis and schizophrenia We report the purification and partial amino-acid sequence from five tryptic fragments of P5 these sequence are not those of any known sequence in the protein identification resource PIR release database synthetic peptide from two of the sequence were used to raise rabbit polyclonal antibodies these antibodies detected P5 on 2DE blots of normal CSF protein and other protein of the same mass with charge distribution between these protein comprise of the total CSF protein and their mass charge abundance and predominance in CSF over plasma are consistent with protein that had been initially characterized with antibodies beta trace protein glycosidase studies confirm that most of these protein are due to sialic acid modifications that are linked to an kDa protein but other charge and mass variations also exist 2DE blots of types of human tissue and body fluid were immunostained Of these anti P5 serum detected protein of the same mass and charge as beta trace protein only in brain samples protein of different mass and charge from beta trace protein were clearly immunostained in samples of eight tissues ABSTRACT TRUNCATED AT WORDS

A freely programmable device for the quantitative post column collection of amino-acid derivatives and elimination products generated by chemical stepwise protein sequence analysis by the edman degadation is described the apparatus is simple can be constructed at low cost from readily available components and can be interfaced with and programmed from common high performance liquid chromatography controllers selected applications illustrate that the device is an essential addition to automated protein sequencers in cases in which chemically or enzymatically modified or metabolically radiolabeled residue are determined at high sensitivities

the transcription termination factor mTERF which plays central role in the control of mitochondrial rRNA and mRNA synthesis in mammalian mitochondria has been previously identified and purified by DNA affinity chromatography from human mitochondrial lysate kruse narasimhan and attardi cell In the present work this factor has been characterized as to its protein composition and the activities of the protein components three polypeptides two of kDa molecular mass and one of kDa were shown to be associated with the specific DNA binding and footprinting activity of the factor with the kDa component having much lower affinity for the recognition sequence than the kDa components On the other hand the transcription termination activity as assayed in an in vitro system was found to be associated exclusively with the two kDa polypeptides mass spectroscopic analysis of tryptic peptide derived from highly purified polypeptides indicated that all three polypeptides share regions with common sequence the evidence obtained suggests that differential phosphorylation is not responsible for the difference in electrophoretic mobility of the three polypeptides

the gene encoding the subunit of DNA polymerase III holoenzyme designated holB was cloned by strategy in which peptide sequence was used to derive DNA hybridization probe the gene maps to centisomes of the chromosome sequencing of holB revealed bp open reading frame predicted to produce Da protein the gene has ribosome binding site and promoter that are highly similar to the consensus sequence and is flanked by two potential open reading frames protein sequence analysis of revealed high degree of similarity to the dnaX gene products of escherichia coli and bacillus subtilis including one stretch of identical amino-acid residue lesser degree of similarity to the gene protein of bacteriophage T4 and the kDa protein of the A1 complex replication factor of HeLa cell was seen the gene when placed into tac promoter based expression plasmid directed expression of two protein of similar size By immunodetection with anti holoenzyme immunoglobulin both protein are judged to be products of holB

We report the design chemical synthesis and structural and functional characterization of novel reagent for protein sequence analysis by the edman degradation yielding amino-acid derivatives rapidly detectable at high sensitivity by ion evaporation mass spectrometry We demonstrate that the reagent ethylene trimethylamino phenyl isothiocyanate is chemically stable and shows coupling and cyclization cleavage yields comparable to phenylisothiocyanate the standard reagent in chemical sequence analysis under conditions typically encountered in manual or automated sequence analysis amino-acid derivatives generated with this reagent were detectable by ion evaporation mass spectrometry at the subfemtomole sensitivity level at pace of one sample per minute furthermore derivatives were identified by their mass thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino-acid derivatives of amino-acid with acidic basic polar or hydrophobic side chains were reproducibly detectable at comparable sensitivities the polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis this reagent used in automated sequence analysis has the potential for overcoming the limitations in sensitivity speed and the ability to characterize modified amino-acid residue inherent in the chemical sequencing method that are currently used

We recently reported the purification of lymphocyte granule protein called fragmentin which was identified as serine protease with the ability to induce oligonucleosomal DNA fragmentation and apoptosis shi kraut aebersold and greenberg exp med We have now purified two additional proteases with fragmentin activity from lymphocyte granules the three proteases are of two types one has the unusual ability to cleave tripeptide thiobenzyl ester substrate after aspartic acid similar to murine cytotoxic cell protease granzyme while two are tryptase like preferentially hydrolyzing after arginine and bear some homology to human cell granule tryptases granzyme and hanukah factor granzyme using tripeptide chloromethyl ketones the pattern of inhibition of DNA fragmentation corresponded to the inhibition of peptide hydrolysis the asp ase fragmentin was blocked by aspartic acid containing tripeptide chloromethyl ketones while the tryptase fragmentins were inhibited by arginine containing chloromethyl ketones the two tryptase fragmentins were slow acting and were partly suppressed by blocking protein synthesis with cycloheximide in the YAC target cell In contrast the asp ase fragmentin was fast acting and produced DNA damage in the absence of protein synthesis using panel of unrelated target cell of lymphoma thymoma and melanoma origin distinct patterns of sensitivity to the three fragmentins were observed thus these three granule proteases make up family of fragmentins that activate DNA fragmentation and apoptosis by acting on unique substrates in different target cell

ISGF is an interferon dependent positive acting transcription factor that is cytoplasmically activated possibly through direct interaction with the interferon receptor the factor has been purified its component protein have been separated and its peptide sequence have been obtained from the sequence degenerate oligonucleotide probes were constructed to screen for cDNA clones sequencing of the selected clones shows that the and kDa components represent two forms of previously unknown to our knowledge protein several antibodies raised against these protein prove that they indeed do encode protein components of ISGF this work provides reagents to explore the modification of this cytoplasmically activated transcription factor

the question of whether protein tyrosine phosphatases PTpases dephosphorylate multiply phosphorylated peptide in random or ordered manner was investigated using the synthetic triphosphotyrosyl peptide TRDIY ETDY RK corresponding to the major sites of autophosphorylation of the insulin receptor as substrate for four purified PTpases all four enzymes dephosphorylated the triphospho peptide to produce diphospho monophospho and nonphosphorylated forms partially dephosphorylated peptide were separated by reverse phase HPLC and the di and monophospho peptide were collected and analyzed by solid phase sequencing to determine the order of dephosphorylation of the three sites by each of the PTpases the quantitative analysis of the signals for derivatives of tyrosine and phosphotyrosine generated at positions and of the peptide showed that the low molecular weight human placental PTpase 1B preferentially dephosphorylated the two phosphotyrosines at positions and whereas the integral membrane enzyme CD45 from human spleen and the bacterially expressed rat LAR preferentially dephosphorylated the phosphotyrosine at position second low molecular weight enzyme termed TCPTpase did not display any specificity for particular phosphotyrosyl residue these results demonstrate that different PTpases exhibit characteristic pattern of dephosphorylation of the triphospho peptide model substrate raising the possibility that features in the primary structure surrounding the dephosphorylation site may contribute to substrate specificity

ISGF is multiprotein transcription factor that is very quickly activated in the cell cytoplasm only after attachment of interferon to the cell surface To understand the specific cytoplasmic activation of protein that move to the nucleus and direct increased transcription of specific gene we have purified and now report completion of the cloning of cDNA encoding the four protein of ISGF with all of the sequence available it is clear that three of these protein are encoded by members of previously unrecognized gene family We suggest that protein encoded by this gene family serve the function of interpreting the fact that cell surface receptor has bound its ligand so that specific signal transduction to the nucleus can occur

alpha interferon stimulates transcription by converting the positive transcriptional regulator ISGF3 from latent to an active form this receptor mediated event occurs in the cytoplasm with subsequent translocation of the activated factor to the nucleus ISGF3 has two components termed ISGF3a and ISGF3 ISGF3 serves as the DNA recognition subunit while ISGF3a which appears to consist of three polypeptides is target for alpha interferon signaling and serves as regulatory component whose activation is required to form ISGF3 ISGF3 DNA binding activity was identified as kDa polypeptide and partial amino-acid sequence has allowed isolation of cDNA clones ISGF3 translated in vitro from recombinant clones bound DNA with specificity indistinguishable from that of ISGF3 purified from HeLa cell sequencing of ISGF3 cDNA clones revealed significant similarity to the interferon regulatory factor IRF family of DNA binding protein in the amino terminal residue of ISGF3 the other IRF family protein bind DNA with specificity related to but distinct from that of ISGF3 We note sequence similarities between the related regions of IRF family protein and the imperfect tryptophan repeats which constitute the DNA binding domain of the myb oncoprotein these sequence similarities suggest that ISGF3 and IRF protein and the myb oncoprotein use common structural motif for DNA recognition recombinant ISGF3 like the natural protein interacted with HeLa cell ISGF3a to form the mature ISGF3 DNA binding complex We suggest that other IRF family members may participate in signaling pathways by interacting with as yet unidentified regulatory subunits analogous to ISGF3a

T cell signaling via the CD4 surface antigen is mediated by the associated tyrosyl protein kinase p56lck the kilodalton mitogen activated protein MAP kinase p42mapk was tyrosyl phosphorylated and activated after treatment of the murine lymphoma cell line 171CD4+ which expresses CD4 with antibody to CD3 treatment of the CD4 deficient cell line with the same antibody did not result in phosphorylation or activation of p42mapk purified p56lck both tyrosyl phosphorylated and stimulated the seryl threonyl phosphotransferase activity of purified p44mpk MAP kinase isoform from sea star oocytes synthetic peptide modeled after the putative regulatory phosphorylation site in murine p42mapk tyr185 was phosphorylated by p56lck with similar vmax but fivefold lower michaelis constant Km than peptide containing the tyr394 autophosphorylation site from p56lck MAP kinases may participate in protein kinase cascades that link src family protein tyrosyl kinases to seryl threonyl kinases such as those encoded by rsk and raf which are putative substrates of MAP kinases

A baculovirus expression system has been used to express large quantities of the lymphocyte specific protein tyrosyl kinase p56lck series of chromatographic steps including the novel application of metalchelate affinity chromatography for protein kinase purification were employed to obtain p56lck in highly active form recombinant p56lck was purified to apparent homogeneity as determined by polyacrylamide gel electrophoretic analyses and was found to migrate in SDS gels as two related species both with apparent molecular masses close to kDa p56lck phosphorylated all assayed substrates exclusively on tyrosyl residue and underwent autophosphorylation at one principal site also on tyrosyl residue p56lck displayed high affinity for synthetic peptide corresponding to the cytoplasmic domain residue of the cell receptor chain TCR Km µM but low affinity for peptide corresponding to its own autophosphorylation site Km µM p56lck was also found to be highly active for purified protein tyrosyl kinase vmax gt pmol min µg using the TCR as substrate variety of agents were tested for their ability to inhibit p56lck with zinc ions I50 mM and staurosporine I50 nM proving the most potent

An intrinsic kDa polypeptide is found associated with the oxygen evolving photosystem II PSII core complex in all green plants and cyanobacteria so far examined although it does not appear to be required for oxygen evolution amino-acid sequence information obtained from the purified kDa protein was used to construct probe that was employed to isolate full length cDNA clone encoding the residue precursor of the kDa protein hydropathy plot analysis predicts the existence of four membrane spanning helices in the mature protein the two halves of the approximately residue mature protein show high sequence similarity to each other suggesting that the psbS gene arose from an internal gene duplication the kDa protein has some sequence similarity to chlorophyll binding protein

We report the purification from rat natural killer RNK large granular lymphocyte leukemia of kD granule protein that induces rapid DNA fragmentation and apoptosis the protein which we have called fragmentin was capable of causing DNA from intact YAC cell to be cleaved into oligonucleosomal sized fragments and producing severe chromatin condensation within amino-acid sequence of tryptic peptide indicated that fragmentin was highly homologous to the NK and cell granule serine proteases RNK protease and mouse cytotoxic cell protease CCPI granzyme preincubation with the serine esterase inhibitor dichloroisocoumarin blocked fragmentin induced DNA damage but had no effect on cytolysin fragmentin activity against four lymphoma target cell was completely dependent on the presence of cytolysin fragmentin produced rapid membrane damage as well as DNA fragmentation at nonlytic cytolysin doses suggesting that fragmentin activity was not limited to its effects on the nucleus fragmentin and cytolysin activity were completely inhibited by EGTA indicating the process was Ca2+ dependent role for cytolysin in endocytosis of fragmentin was suggested by the observation that treatment of YAC with cytochalasin or sodium azide and deoxyglucose blocked DNA fragmentation but not cytolysin activity kD Na CBZ lysine thiobenzyl esterase which copurified with fragmentin was inactive on its own but was able to synergistically amplify the DNA damage induced by fragmentin in the presence of cytolysin fragmentin activity was not dependent on protein synthesis as cycloheximide treatment of YAC cell did not prevent DNA damage We postulate that fragmentin is the molecular mediator of NK cell mediated DNA fragmentation and apoptosis

using an oligonucleotide hybridization probe we have mapped the structural gene for the subunit of escherichia coli DNA polymerase III holoenzyme to centisomes of the chromosome this gene designated holA was cloned and sequenced the sequence of holA matches precisely four amino-acid sequence obtained for the amino terminus of and three internal tryptic peptide holA overproducing plasmid that directs the expression of up to of the soluble protein was constructed sequence analysis of holA revealed bp open reading frame that encodes protein with predicted molecular mass of Da holA may reside downstream of rlpB in an operon perhaps representing yet another link between structural gene for the DNA polymerase III holoenzyme and protein involved in membrane biogenesis these and other features are discussed in terms of genetic regulation of subunit synthesis

the covalent intermediate formed during catalysis by the lac galactosidase from escherichia coli can be trapped by reaction of the enzyme with dinitrophenyl deoxy fluoro galactopyranoside thereby inactivating the enzyme kinetic parameters for this inactivation process with the holo and apoenzymes have been determined the intermediate so formed turns over only very slowly resulting in reactivation of the enzyme the nucleophilic amino-acid involved has been identified as glu by using tritium labeled inactivator to label the enzyme then cleaving the labeled protein into peptide and purifying and sequencing the labeled peptide this residue is conserved in five homologous galactosidases and is different from that glu proposed to be the nucleophile herrchen and legler eur biochem on the basis of affinity labeling studies with conduritol cis epoxide role for glutamic acid residue as the acid base catalyst is proposed and justified

A type II DNA topoisomerase topoIimt was shown previously to be associated with the kinetoplast DNA of the trypanosomatid crithidia fasciculata the gene encoding this kinetoplast associated topoisomerase has been cloned by immunological screening of crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme the gene cfaTOP2 is single copy gene and is expressed as kb polyadenylated transcript the nucleotide sequence of cfaTOP2 has been determined and encodes predicted polypeptide of amino-acid with molecular mass of the identification of the cloned gene is supported by immunoblot analysis of the galactosidase cfaTOP2 fusion protein expressed in escherichia coli and by analysis of tryptic peptide sequence derived from purified topoIimt cfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene

using an improved SDS PAGE system the polypeptides of the major chlorophyll light harvesting complex of PSII LHCII from tomato leaves were resolved into five polypeptide bands all the polypeptides were matched with the gene encoding them by comparing amino-acid sequence of tryptic peptide with gene sequence the two major LHCII bands usually comigrating as kDa polypeptide were encoded by cab1 and cab3 type LHCII gene third strong band of about kDa was encoded by cab4 type II gene polypeptides from two minor bands of kDa were not terminally blocked their terminal sequence showed they were type III LHCII protein one complete cDNA clone and several incomplete clones for type III polypeptides were sequenced combined with the peptide sequence the results indicate that there are at least four different type III gene in tomato encoding four almost identical polypeptides thus all the LHCII CAB polypeptides have been identified and each type of LHCII polypeptide is encoded by distinct gene or gene in tomato

In this report we describe the use of novel experimental polyvinylidene fluoride based membrane with cationic surface for the isolation by electroblotting of small amounts of protein separated by gel electrophoresis for further characterization by protein fragmentation for internal sequence analysis the membrane is characterized by surface that mediates primarily ionic protein membrane interactions and that allows the recovery of adsorbed protein at high yields under relatively mild conditions In electroblotting experiments the novel membrane has binding capacity that is at least equivalent to that of standard polyvinylidene fluoride membranes and is compatible with both chemical and enzymatic fragmentation of blotted protein in situ intact electroblotted protein or fragments thereof were eluted at high yields further structural analysis is demonstrated using reverse phase high performance liquid chromatography or gel electrophoresis to separate cleavage fragments for either pulsed liquid or solid phase automated sequence analysis

A method for the determination of the sites of tyrosine phosphorylation in protein and peptide at the low picomole level for cold phosphopeptides and at the subpicomole level for 32P labeled phosphopeptides is presented the procedure is based on solid phase sequence analysis of phosphopeptides immobilized on carrier discs and the on line detection by reverse phase high performance liquid chromatography of the phenylthiohydantoin derivative of phosphotyrosine the procedure is sensitive and automated and allows the identification of phosphotyrosine derivatives in the same operation as the detection of the derivatives of the other common amino-acid essentially quantitative extraction of the phosphotyrosine derivatives from the sequencer makes this method ideally suited for the quantitative assessment of protein tyrosine kinase and protein phosphatase activities and for the determination of their respective recognition sequence

cenA and cex are glycanases produced by the cellulolytic bacterium cellulomonas fimi both enzymes are composed of two domains and contain six cys residue two disulfide bonds were assigned in both enzymes by peptide analysis of the isolated catalytic domains further disulfide bond was deduced in both cellulose binding domains from the absence of free thiols under denaturing conditions corresponding cys residue are conserved in eight of nine other known fimi type cellulose binding domains cenA and cex belong to families and respectively in the classification of glucanases and xylanases based on similarities in catalytic domain primary structure disulfide bonds in the cenA catalytic domain correspond to the two disulfide bonds in the catalytic domain of trichoderma reesei cellobiohydrolase II family which stabilize loops forming the active site tunnel sequence alignment indicates the probable occurrence of disulfides at equivalent positions in the two other family enzymes partial resequencing of the gene encoding streptomyces KSM glucanase casA family revealed five errors in the original nucleotide sequence analysis the corrected amino-acid sequence contains an asp residue corresponding to the proposed proton donor in hydrolysis catalysed by cellobiohydrolase II cys residue which form disulfide bonds in the cex catalytic domain are conserved in xynZ of clostridium thermocellum and xyn of cryptococcus albidus but not in the other eight known family enzymes like other members of its family cex catalyses xylan hydrolysis the catalytic efficiency cat for hydrolysis of the heterosidic bond of nitrophenyl xylobioside is min mM at the corresponding cat for nitrophenyl cellobioside hydrolysis is min mM

A sensitive automated and nonisotopic assay for protein tyrosine kinases and phosphatases has been developed the assay uses commercially available antiphosphotyrosine monoclonal antibodies and the recently developed particle concentration immunofluorescence immunoassay technology the assay is specific for phosphotyrosine residue can be performed faster and is at least fold more sensitive than the current standard filter type radioassay myelin basic protein and synthetic peptide corresponding to the autophosphorylation site of p56lck performed equally well in the detection of p56lck kinase activity myelin basic protein phosphorylated on tyrosine residue by p56lck was successfully used as substrate in the detection of phosphatase activity and vanadate or molybdate were shown to inhibit the phosphatase activity the assay is particularly useful for the rapid detection of enzyme activities in column fractions from biochemical procedures steps and also for screening of large numbers of potential inhibitors or activators of protein tyrosine kinases and phosphatases

In addition to its known substrate activity with pnitrophenyl cellobioside the exoglucanase from cellulomonas fimi has been shown to utilize substituted phenyl glucosides as substrates of which the best is dinitrophenyl glucopyranoside the enzyme can be inactivated by treatment with dinitrophenyl deoxy fluoro glucopyranoside by trapping of the covalent intermediate in catalysis as has been shown for glucosidase withers and street Am chem soc the intermediate formed is stable but can undergo turnover in the presence of cellobiose reactivating the enzyme by transglycosylation using tritium labeled inactivator it has been possible to isolate and sequence radiolabeled peptide from this enzyme and the active site nucleophile has been identified as glutamic acid residue this glutamic acid residue and its sequentially proximal amino-acid are absolutely conserved in the homologous family of cellulases

two recently identified pro inflammatory protein namely neutrophil activating peptide NAP also termed interleukin IL and NAP were chemically synthesized purified and characterized the fully protected NAP IL residue and NAP residue peptide chains were assembled by automated solid phase method with average stepwise yields of and resulting in overall chain assembly yields of and respectively deprotection resulted in crude products which were allowed to fold by air oxidation and were purified by two cycles of reverse phase high pressure liquid chromatography yielding mg of NAP IL and mg of NAP purity was established by reverse phase high pressure liquid chromatography and isoelectric focusing and the primary structures of the purified products were verified by using mass spectrometry and edman sequencing method synthetic and recombinant NAP IL were equally active on human neutrophil granulocytes as determined by measuring the induction of cytosolic free calcium elastase release and chemotaxis synthetic NAP was equivalent to purified natural NAP in the elastase release and calcium mobilization assay but it was consistently less potent fold as stimulus of chemotaxis perhaps indicative of additional chemotactic components in the natural preparation the results indicate that by chemical synthesis these cytokines can be obtained in purity and quantities suitable for further structural analysis as well as functional studies both in vivo and in vitro the ability to rapidly generate analogues with unambiguous primary structure suggests that this will be the method of choice for an in depth study of structure function relationships within this family of inflammatory cytokines

the Da protein pp160 which is rapidly phosphorylated on tyrosine in response to insulin and thus is putative participant in signaling from the insulin receptor has been purified to homogeneity from 3T3 L1 adipocytes isolation of this protein was accomplished by chromatography on an immobilized monoclonal antibody against phosphotyrosine followed by gel electrophoresis sufficient protein was obtained to allow the determination of the sequence of several peptide which in turn enabled the development of anti peptide antibodies that specifically recognize pp160 immunoblotting of 3T3 L1 adipocyte lysates together with the purified pp160 as standard indicate that an insulin treated 3T3 L1 adipocyte possesses about copies of tyrosine phosphoryl ated pp160 and that this amount is approximately of the total pp160 in the cell the number of tyrosine phosphorylated pp160s per cell is approximately the same as that of insulin receptor subunits these results provide further evidence for role of pp160 in insulin signaling moreover the availability of purified protein and knowledge of peptide sequence will allow the elucidation of the structure and function of this protein

A yeast membrane protein was isolated by its binding to tRNA sepharose column the kDa protein shares characteristics with rat liver nuclear pore protein in having reactivity with monoclonal antibody RL1 raised against rat liver nuclear pore protein and by the bindung of wheat germ agglutinin WGA indicating the presence of acetylglucosamine glcNAc moieties immunofluorescence microscopy and cell fractionation experiments indicate that the protein is located in the nuclear envelope and the endoplasmic reticulum of the cell the gene for the kDa protein was cloned using degenerate oligonucleotides derived from the terminal protein sequence and confirmed by internal peptide sequence the gene was named WBP1 the protein coding sequence of the WBPl gene reveals an ER entry signal peptide and terminal membrane spanning domain topological studies indicate that the terminus of the protein is located in the cytoplasm the cytoplasmic tail of the protein contains the signal known to be sufficient for retention of transmembrane protein in higher eukaryotic cell gene disruption experiments show that the kDa protein is essential for the vegetative life cycle of the yeast cell

We have identified by combination of ligand 45Ca2+ and immunoblotting two large membrane protein akin to the mammalian so called low density lipoprotein LDL receptor related protein LRP in chicken tissues LRP has thus far been demonstrated only in mammalian species where it is thought to act as receptor for proteinase a2 macroglobulin complexes and or chylomicron remnants lipoproteins not produced in birds one of the chicken LRPs was demonstrated in liver and has the same apparent and hallmark biochemical properties as rat liver LRP the other chicken LRP is smaller kDa and is expressed in ovarian follicles but is undetectable in liver immunological analysis demonstrated lack of cross reactivity between the two LRPs as well as between them and the previously identified chicken oocyte specific kDa receptor for the yolk precursors very low density lipoprotein and vitellogenin stifani barber nimpf and schneider proc natl acad sci As shown by ligand blotting both chicken LRPs have the ability to interact with vitellogenin property they share not only with rat LRP but also with mammalian LDL receptors To obtain independent conformation of the ligand blotting results the smaller follicular LRP was purified and high affinity binding of vitellogenin to it was demonstrated by solid phase filtration binding assay amino-acid sequence of tryptic fragments of the smaller LRP were obtained and its homology with human LRP demonstrated through unambiguous alignment of three fragments both chicken LRPs the chicken oocyte kDa receptor as well as rat LRP could be shown by ligand blotting to interact specifically with chicken serum a2 macroglobulin In addition human apolipoprotein ligand implicated in receptor mediated metabolism of chylomicron remnants also binds to the smaller chicken LRP further emphasizing the similarities between LDL receptors and related protein from variety of species In analogy to the known dichotomy of chicken LDL receptors which is characterized by the production of the kDa oocyte specific receptor on one hand and kDa LDL receptor that is exclusively expressed in somatic cell hayashi nimpf and schneider biol chem it appears that the smaller and larger chicken LRPs also may be restricted to the oocyte and somatic cell respectively

the final rapid growth phase of the chicken oocyte is characterized by massive uptake of hepatically syn thesized yolk precursor protein from the plasma the two major yolk forming components very low density lipoprotein VLDL and vitellogenin VTG have been shown to interact with kDa protein present in detergent extracts of ovarian membranes this protein is absent in hens of mutant nonlaying chicken strain nimpf radosavljevic and schneider biol chem here we have purified the kDa protein by ligand and immunoaffinity chromatography and demonstrated its role in receptor mediated endocytosis by ultrastructural immunolocalization structural and functional studies the receptor was visualized exclusively in the oocyte proper and was absent from somatic cell in agreement with the previously reported expression of two different lipoprotein receptors in somatic cell and oocytes respectively of laying hens hayashi nimpf and schneider biol chem amino-acid sequence of tryptic fragments of the oocyte receptor were obtained and its kinship to somatic low density lipoprotein receptors was confirmed through the demonstration of sequence conservation in three characteristic domains In particular the chicken receptor internalization sequence pheasp asn pro val tyr is identical with that in low density lipoprotein receptors from mammals as well as xenopus laevis the ligand binding properties specificity and kinetic parameters of the oocyte receptor were characterized in filtration assay employing pure ligands and receptor In conjunction with ligand blotting experiments following limited protease digestion of the receptor the binding assay data suggest that VTG recognizes substructure of the VLDL binding site these studies establish that cell specific receptor mediates the endocytosis of VTG and VLDL into growing chicken oocytes and thus possibly plays key role in control of oocyte growth

CP29 the core chlorophyll CAB antenna complex of photosystem II PSII has two nuclearencoded polypeptides of approximately and kDa in tomato lycopersicon esculentum cab9 the gene for the type kDa CP29 polypeptide was cloned by immunoscreening tomato leaf cDNA library its identity was confirmed by sequencing tryptic peptide from the mature protein cab9 is single copy gene with five introns the highest number found in CAB protein In vitro transcription translation gave kDa precursor which was cleaved to about kDa after import into isolated tomato chloroplasts the cab9 polypeptide has the two highly conserved regions common to all CAB polypeptides which define the members of this extended gene family outside of the conserved regions it is only slightly more closely related to other PSII CABs than to PSI CABs sequence analysis of tryptic peptide from the type II kDa CP29 polypeptide showed that it is also member of the CAB family and is very similar or identical to the CP29 polypeptide previously isolated from spinach all members of the CAB family have absolutely conserved his gln and asn residue which could ligate the Mg atoms of the chlorophylls and number of conserved asp glu lys and arg residue which could form bonds to the polar groups on the porphyrin rings the two conserved regions comprise the first and third predicted trans membrane helices and the stroma exposed segments preceding them

A gt10 bovine brain and gt11 bovine heart cDNA library were screened with oligonucleotide probes corresponding to partial protein sequence directly determined from the isolated kDa subunit of the bovine respiratory chain NADH dehydrogenase clones were isolated that encode protein of amino-acid containing all the partial tryptic peptide sequence determined from the kDa subunit the size and amino-acid composition of this protein agree with those determined for the purified kDa subunit furthermore this protein contains putative NADH binding domain possible FMN binding site and putative binding site for an iron sulfur cluster the above evidence indicates that the cloned protein is the kDa subunit or its precursor search for sequence similarity with protein in the protein identification resource data base has revealed that the kDa subunit has amino-acid sequence identity with major portion of the subunit of the soluble NAD+ reducing hydrogenase from alcaligenes eutrophus In particular there are three segments of high sequence similarity between the two protein which correspond to the three ligand binding sites

meiotic maturation of xenopus and sea star oocytes involves the activation of number of protein serine threonine kinase activities including myelin basic protein MBP kinase kDa MBP kinase p44mpk purified from mature sea star oocytes is shown here to be phosphorylated at tyrosine antiserum to purified sea star p44mpk was used to identify antigenically related protein in xenopus oocytes two tyrosine phosphorylated kDa protein p42 were detected with this antiserum in xenopus eggs xenopus p42 chromatographs with MBP kinase activity on mono ion exchange column tyrosine phosphorylation of xenopus p42 approximately parallels MBP kinase activity during meiotic maturation these results suggest that related MBP kinases are activated during meiotic maturation of xenopus and sea star oocytes previous studies have suggested that xenopus p42 is related to the mitogen activated protein MAP kinases of cultured mammalian cell We have cloned MAP kinase relative from xenopus ovary cDNA library and demonstrate that this clone encodes the xenopus p42 that is tyrosine phosphorylated during oocyte maturation comparison of the sequence of xenopus p42 and rat MAP kinase ERK1 and peptide sequence from sea star p44mpk indicates that these protein are close relatives the family members appear to be tyrosine phosphorylated and activated in different contexts with the murine MAP kinase active during the transition from quiescence to the G1 stage of the mitotic cell cycle and the sea star and xenopus kinases being active during phase of the meiotic cell cycle

methods were developed for high yield covalent attachment of peptide and protein to isothiocyanate and arylamine derivatized poly vinylidene difluoride membranes for solid phase sequence analysis solutions of protein or peptide were dried onto mm membrane disks such that the functional groups on the surface and the polypeptide were brought into close proximity In the case of the isothiocyanate membrane reaction between polypeptide amino groups and the surface isothiocyanate moieties was promoted by application of aqueous methylmorpholine attachment of protein and peptide to the arylamine surface was achieved by application of water soluble carbodiimide in pH buffer edman degradation of covalently bound polypeptides was accomplished with initial and repetitive sequence yields ranging from to and to respectively the yields were independent of the sample load pmol to nmol for either surface significant loss of material was not observed when attachment residue were encountered during sequence runs application of bovine lactoglobulin chain staphylococcus protein or the peptide melittin to the isothiocyanate membrane allowed for extended terminal sequence identification residue from pmol of lactoglobulin number of synthetic and naturally occurring peptide were sequenced to the terminal residue following attachment to the arylamine surface In one example µg of bovine casein was digested with staphylococcal protease V8 and the peptide were separated by reverse phase chromatography peptide fractions were then directly applied to arylamine membrane disks for covalent sequence analysis from as little as pmol of initial signal it was possible to determine substantial sequence information residue

tryptic peptide sequence from the kDa polypeptide of tomato LHCI were used to construct probe for gene cloning the two gene cloned cab11 and cab12 encode protein of and residue that are identical in overall amino-acid sequence and identical in the deduced mature protein each gene is present in single copy per haploid genome cab11 on chromosome and cab12 on chromosome and each has introns located in similar positions to introns in other members of the chl binding CAB protein gene family comparison of the amino-acid sequence of LHCI LHCII CP29 and CP24 polypeptides confirms that all CABs share two regions of very high similarity which include the first and third transmembrane helices and the stroma exposed sequence preceding them however near the terminus and between the conserved regions the LHCI polypeptides have sequence motifs which appear to be PSI specific

the solutions to many protein analytical problems require flexible chemistries at high sensitivity solid phase sequence analysis is one way to eliminate limitations inherent in current method

plastins are family of at least three cytoplasmic protein isoforms that are expressed differentially between cell of the hematopoietic lineages and cell of solid tissues expression of the plastin isoform appears to be restricted to replicating blood cell and the two plastin isoforms appear to be restricted to replicating cell of solid tissues however plastin is induced in many human solid tumor derived cell We used the anchored polymerase chain reaction technique to amplify and clone the missing ends of plastin mRNAs We found that both plastin isoforms contain potential calcium binding site near the terminus

myelin basic protein serves as convenient substrate for detection of kDa protein serine threonine kinase p44mpk that is activated near the time of germinal vesicle breakdown in maturing echinoderm and amphibian oocytes In vitro phosphorylation by purified p44mpk from sea star oocytes was primarily on threonine residue on single tryptic peptide of bovine brain myelin basic protein amino-acid composition analysis of the isolated posphopeptide revealed that it was rich in proline residue automated solid phase sequencing by edman degradation identified the major site as thr in the sequence NIVTPRTPPPSQGK which corresponds to residue in bovine brain myelin basic protein thr was also phosphorylated by p44mpk to very minor extent

We report method for the analysis of dilute peptide solutions by capillary zone electrophoresis the procedure is based on an electrophoretic concentration step of the applied peptide solution in the capillary stacking prior to separation thus allowing the application of increased sample volumes without breakdown in resolution given constant configuration of the hardware the method permits the analysis of peptide solutions of an at least times lower concentration than previously possible the method was applied to the direct analysis of peptide samples separated by narrow bore reversed phase high performance liquid chromatography for high sensitivity peptide sequence analysis

improved technologies or the synergistic use of complementary method enhance the efficiency of research and permit the exploration of new approaches for the investigation of complex problems high sensitivity protein sequence analysis and polyacrylamide gel electrophoresis are such complementary method here we summarize the current status of high sensitivity sequence analysis of protein separated in polyacrylamide gels and discuss strategies by which this technology can enhance biological research by generating new approaches for the solution of complex multifacetted problems finally we outline imminent technological advances in the area of high sensitivity protein sequence analysis and argue that further technological developments will ultimately lead to the generation of an integrated protein database containing structural and functional as well as physiological information in an easily accessible form of all the protein separated by high resolution two dimensional gel electrophoresis

CTP phosphocholine cytidylyltransferase EC is key regulatory enzyme in the synthesis of phosphatidylcholine in higher eukaryotes this enzyme can interconvert between an inactive cytosolic form and an active membrane bound form To unravel the structure of the transferase and the mechanism of its interaction with membranes we have cloned cytidylyltransferase cDNA from rat liver by the oligonucleotide directed polymerase chain reaction transfection of the rat clone into COS cell resulted in fold increase in cytidylyltransferase activity and content the activity of the transfected transferase was lipid dependent the central portion of the derived protein sequence of the rat clone is highly homologous to the previously determined yeast cytidylyltransferase sequence tsukagoshi nikawa and yamashita eur biochem the rat protein sequence lacks any signals for covalent lipid attachment and lacks hydrophobic domain long enough to span bilayer however it does contain potential residue amphipathic helix encompassing three homologous residue repeats We propose that the interaction of cytidylyltransferase with membranes is mediated by this amphipathic helix lying on the surface with its axis parallel to the plane of the membrane such that its hydrophobic residue intercalate the phospholipids

We have developed method for the high efficiency covalent immobilization of picomole to nanomole quantities of peptide in form compatible with high sensitivity gas liquid or solid phase sequence analysis glass fiber filter paper was derivatized with aminophenyltriethoxysilane and peptide were applied to circular disks cut to cm diameters peptide were covalently immobilized on the aminophenyl glass fiber paper through their terminal carboxyl groups and amino-acid side chain carboxyl groups by activation with the water soluble reagent ethyl dimethylaminopropyl carbodiimide disks containing the covalently attached peptide were directly inserted into the cartridge of an automated sequenator for sequence analysis by the edman degradation peptide prepared in this way could be routinely sequenced through to and including the terminal amino-acid residue at extraordinarily low backgrounds the covalent immobilization of peptide fragments allowed far more flexibility in sequencing conditions including the use of polar extraction solvents to increase the yield of phenylthiohydantoin PTH his and PTH arg and the use of alterantive edman type sequencing reagents with enhanced detectability such as the chromophoric compound dimethylamino azobenzene isothiocyanate the potential of this high efficiency immobilization method for contributing to the development of sequencing chemistries with enhanced sensitivity is discussed

A protein secreted by cultured rat heart cell can direct the choice of neurotransmitter phenotype made by cultured rat sympathetic neurons structural analysis and biological assay demonstrated that this protein is identical to protein that regulates the growth and differentiation of embryonic stem cell and myeloid cell and that stimulates bone remodeling and acute phase protein synthesis in hepatocytes this protein has been termed factor DIA DIF DRF HSFIII and LIF thus this cytokine like IL and TGFß regulates growth and differentiation in the embryo and in the adult in many tissues now including the nervous system

guanine nucleotide binding protein protein that transduce signals from cell surface receptors to effector molecules are made up of three subunits and complementary DNA clone that encodes amino-acid protein was isolated from bovine brain this protein contains peptide sequence that were derived from the purified subunit of and the primary sequence of this protein subunit has percent homology to the subunit of transducin and also has homology to functional domains of mammalian ras protein the probe for isolating the clone was generated with the use of the polymerase chain reaction PCR the extent of divergence between and the isolation of homologous PCR generated fragments and the differences between the predicted amino-acid sequence of and that derived from the subunit of and indicate that subunits are encoded by family of gene

large arteries have natural resistance to tumor cell invasion thought to be due to the production of protease inhibitors vascular smooth muscle cell VSMC representing the major cellular part of arteries were isolated from human aortas and grown in tissue culture these cell were found to produce large amounts of inhibitors of plasminogen activators PA fractionation of VSMC conditioned medium by heparin affigel chromatography separated three immunologically and functionally distinct PA inhibitors PAI namely PAI PAI and protease nexin the three inhibitors were characterized by functional assay and immunoblotting PA inhibitor PAI had little affinity for heparin whereas PA inhibitor PAI bound to heparin and was eluted from the column at NaCl concentrations of to protease nexin eluted at NaCl concentrations of and higher most of the PAI was present in the latent inactive form PAI was further purified by ion exchange chromatography on mono column partial sequencing of the purified PAI confirmed its nature by matching completely with the sequence deduced from the cDNA nucleotide sequence of endothelial cell PAI thus human VSMC produce all three presently known PAI and these can be separated in single heparin affinity purification step

We report new method for the preparation of protein in form suitable for high sensitivity terminal amino-acid sequence analysis protein separated by polyacrylamide gel electrophoresis were electrophoretically transferred onto glass fiber filter paper chemically activated by the introduction of phenyl isothiocyanate functional groups the protein became covalently coupled to the matrix during the electrotransfer process bands containing transferred protein were detected by fluorescent staining or autoradiography cut out from the glass fiber filter and directly loaded into the cartridge of gas phase sequenator the covalent nature of the interactions between protein and glass fiber support permitted the use of more vigorous solid phase sequencing protocols and of alternative sequencing reagents this high efficiency isolation and covalent coupling method provides the essential first step toward enhanced sensitivity protein sequence analysis the method has been successfully applied to the isolation of wide variety of protein from SDS polyacrylamide gels and was shown to be compatible with both the standard edman reagent phenyl isothiocyanate and alternative sequencing reagents such as dimethylamino azobenzene isothiocyanate DABITC

the memory response of BALB mice to phosphocholine keyhole limpet haemocyanin PC KLH consists of two antibody populations designated group and group II that differ in their fine specificity as determined by hapten inhibition using phosphocholine PC and nitrophenylphosphocholine NPPC It is shown that group response is dominated by T15 idiotype positive antibodies that utilize the VH1 heavy chain gene in combination with light chain gene In this paper we show that group II serum antibodies of BALB mice are also highly restricted in their heterogeneity as determined by terminal amino-acid sequence analysis furthermore we demonstrate that the group II response is not affected by neonatally induced T15 suppression whereas the group response in these mice consists of T15 negative antibodies this suggests that the expression of the two antibody populations is regulated independently finally we show that the isotype distributions within fine specificity are the same in normal and T15 suppressed mice interestingly this is true not only for the group II but also for the group antibodies because the isolated group antibodies from normal mice differ in structure from those of T15 suppressed mice different light chains our data indicate that the isotype distribution of these two populations is associated with their fine specificity in addition to their clonal origin

We have developed general two step method for obtaining peptide fragments for sequence analysis from picomole quantities of protein separated by gel electrophoresis after separation by one or two dimensional polyacrylamide gel electrophoresis protein are electrophoretically transferred electroblotted onto nitrocellulose the protein containing regions are detected by reversible staining and are cut out and each protein is digested in situ by proteolytic enzymes such as trypsin or staphylococcal protease the resulting peptide fragments are separated by narrow bore reverse phase HPLC collected and sequenced in gas phase sequenator excellent peptide recoveries and the absence of extraneous contaminants in the separation of the peptide fragment mixture allow the generation of extensive internal sequence information from picomole amounts of protein the method thus overcomes the problem of obtaining amino-acid sequence data from terminally blocked protein and provides multiple independent stretches of sequence that can be used to generate oligonucleotide probes for molecular cloning and or used to search sequence data bases for related protein this method has been successfully applied to the routine amino-acid sequence analysis of wide range of protein isolated from one and two dimensional polyacrylamide gels

the drosophila position specific PS antigens are family of cell surface glycoprotein complexes thought to be involved in morphogenesis their overall structures and biochemical properties are similar to those of group of vertebrate receptors including those for fibronectin fibrinogen and vitronectin and also the leukocyte antigens mac LFA and p150 and the VLA family of cell surface antigens the terminal sequence of the alpha subunits of some of these molecules are homologous to the terminus of PS antigen component the drosophila PS antigens thus appear to be homologous to these vertebrate receptors

We have developed new method for the isolation of protein for microsequencing It consists of electrophoretic transfer electroblotting of protein or their cleavage fragments onto activated glass filter paper sheets immediately after separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis the protein are immobilized on the glass fiber sheets by ionic interactions or by covalent attachment wide range of protein can be prepared in this fashion with no apparent restriction due to solubility size charge or other intrinsic properties of the protein As little as ng of the transferred protein can be detected using coomassie blue or fluorescent dye staining procedures and even smaller amounts of radiolabeled protein by autoradiography after detection the protein containing bands or spots are cut out and inserted directly into gas phase sequenator the piece of glass fiber sheet acts as support for the protein during the sequencing amounts of protein in the to pmol range can be sequenced and extended runs can be obtained from the blotted samples because of improved stepwise yields and lower backgrounds the method has been successfully applied to the sequencing of variety of protein and peptide isolated from one dimensional and two dimensional polyacrylamide gels

general method for the study of the primary structure of picomole quantities of large hydrophobic membrane glycoproteins with blocked amino termini have been developed three techniques designed to be used in concert with each other are described first modified protein preparation and fragmentation techniques secondly simple but very selective two dimensional reversed phase high performance liquid chromatography system for the resolution of complex mixtures of small to medium sized tryptic peptide on vydac C4 C18 and diphenyl columns and thirdly two dimensional separation method for large denaturated CNBr polypeptide fragments by size exclusion high performance liquid chromatography combined with either reversed phase high performance liquid chromatography C4 or sodium dodecyl sulphate polyacrylamide gel electrophoresis in conjunction with electroblotting and autoradiography these method were applied to studies of the platelet derived growth factor receptor starting with pmoles of purified protein total of amino-acid were sequenced

the first complete amino-acid sequence of the variable region of the gamma heavy chain from murine anti streptococcal group polysaccharide CHO immunoglobulin monoclonal antibody 2S1 is described therefore in conjunction with the previously published 2S1 light chain sequence kappa structure the entire variable domain of this antibody has been determined In addition four partial amino terminal heavy chain sequence of other antibodies with the same specificity are reported these heavy chains share high degree of homology with heavy chains from fructosan binding murine myeloma protein with the exception of those positions known to be encoded by the diversity segment in germ line DNA the light chains associated with the heavy chains reported here are products of the kappa kappa and kappa gene Up to date three VH and four kappa subgroups have been shown to contribute genetic material to the assembly of antibodies specific for the CHO unlike other experimental system employing structurally completely resolved full antigens the antistreptococcal immune response uses gene previously shown to be involved in the formation of antibodies with different specificities this provides further experimental evidence for the physiological relevance of heavy light chain association as posttranscriptional diversification mechanism in the generation of the antibody repertoire in addition to those somatic diversifiers acting directly upon the gene encoding the variable regions of individual chains

three murine anti phosphorylcholine PC hybridomas with group II like fine specificity patterns isolated during memory response to PC keyhole limpet hemocyanin KLH are examined at the molecular level to determine the origins of the and used by these antibodies southern blots of hind III cut DNA were hybridized with probe specific for the V1 gene of the T15 family the V1 germ line configuration is retained in these hybridomas indicating that this gene which encodes the gene product expressed by most group anti PC hybridomas is not used for antibody production southern blots of eco RI cut DNA hybridized to probe specific for indicated that all three hybridomas PCG1 PCG1 and PCM share kb rearranged band suggesting utilization of common gene segment terminal amino-acid sequence analysis of the heavy chains of two of the hybridoma protein PCG1 and PCG1 indicates that they belong to mouse heavy chain subgroup II and are closest in sequence to isotype anti PC hybridoma protein HPC derived from BALB mice suppressed for the T15 idiotype PCG1 and PCG1 each differed from HPC at only residue In addition these protein have in common lysine at position which has not been found previously in other heavy chain sequence reported terminal sequence of the light chains of PCG1 and PCG1 are each shown to differ at only residue from and PCM had previously been shown to use both of these light chains have been associated previously with heavy chains derived from the V1 gene in anti PC antibodies these results indicate that the isotype can be used during normal antibody response to PC and thus that heavy chains derived from both subgroup II and subgroup III the T15 heavy chain contribute to the molecular heterogeneity observed in memory responses to PC KLH

specific photoaffinity labelling of purified electric eel acetylcholinesterase by 3H labelled dimethyl amino benzenediazonium fluoroborate allows the identification of labelled peptide fragment which is described as being involved in the binding of quaternary ammonium ions on this enzyme denaturation and proteolytic cleavage of the inactivated enzyme gave mixture of peptide fragments the purification of one labelled fragment containing over of the radioactivity incorporated in the enzyme led to the following sequence gly ser phe the relatively low amount of this tetrapeptide did not allow us to determine the nature of the labelled residue

interleukin IL protein of amino-acid was chemically synthesized by means of an automated peptide synthesizer and was shown to have the biological activities attributed to native IL assay of synthetic analogues established that an amino terminal fragment has detectable Il activity but that the stable tertiary structure of the complete molecule was required for full activity the results demonstrate that automated peptide synthesis can be applied to the study of the structure and function of protein

partial terminal amino-acid sequence for the three largest nonstructural protein of two flaviviruses yellow fever virus and St louis encephalitis virus have been obtained the determined sequence of these protein exhibit significant amino-acid sequence homology and allow the positioning of these three nonstructural protein in the polyprotein sequence deduced from the nucleotide sequence of yellow fever virus rice lenches eddy shin sheets and strauss science229 the deduced start points support the hypothesis that the terminus of nonstructural glycoprotein NS1 results from cleavage by signalase whereas the termini of NS3 and NS5 result from cleavages following double basic residue that are flanked by amino-acid with short side chains

A clone encoding PrP the major protein in purified preparations of scrapie agent was selected from scrapie infected hamster brain cDNA library by oligo nucleotide probes corresponding to the terminus of the protein southern blotting with PrP cDNA revealed single gene with the same restriction patterns in normal and acrapie infected brain DNA single PrP related gene was also detected in murine and human DNA PrP related mRNA was found at similar levels in normal and scrapie infected hamster brain as well as in many other normal tissues using antisera against PrP PrP related protein was detected in crude extracts of infected brain and to lesser extent in extracts of normal brain proteinase digestion yielded PrP in infected brain extract but completely degraded the PrP related protein in normal brain extract No PrP related nucleic acids were found in purified preparations of scrapie prions indicating that PrP is not encoded by nucleic acid carried within the infectious particles

antibody 50S10 is hybridoma derived gamma kappa antibody of BAB mouse strain origin with specificity for acetylglucosamine beta linked to rhamnose the immunodeterminant of the streptococcal group polysaccharide the VL50S10 amino-acid sequence is the fourth complete one reported with this specificity and the first fully determined kappa 21A structure furthermore it is the first kappa 21A isotype sequence derived from an antibody with known antigen specificity the kappa region of this and the previously described monoclonal anti streptococcal group polysaccharide antibodies 7S34 2S1 and 17S29 are compared showing that in monoclonal antibody 50S10 kappa germline gene is expressed which is unrelated to those previously shown to be expressed in antibodies of this specificity kappa 50S10 increases the variability of known murine kappa regions and confirms stretches of kappa 21A sequence previously established

antibodies 17S29 and 22S25 are monoclonal hybridoma derived gamma kappa murine immunoglobulins with specificity for acetyl glucosamine beta linked to the rhamnose backbone structure the immunodeterminant of the streptococcal group polysaccharide the VL 17S29 amino-acid sequence is the third complete one reported from an antibody with this specificity the second fully determined kappa structure and the first complete kappa sequence of C57B1 origin derived from carbohydrate specific antibody VL22S25 is member of the kappa isotype of murine immunoglobulin VL regions kappa 17S29 and the determined part of the kappa 22S25 sequence are compared to the previously described kappa regions of streptococcal group polysaccharide specific antibodies and to selected partial and complete kappa regions of antibodies with other specificities predominantly to carbohydrate antigens both kappa 17S29 and kappa 22S25 increase the variability of known murine kappa regions they are the most homologous to the other kappa regions derived from antibodies with streptococcal group polysaccharide specificity and share with them the amino-acid residue arg74 so far characteristic for kappa regions from antibodies with this specificity the analysis of groups of independently expressed highly homologous kappa regions namely kappa 17S29 and kappa 2S1 as one and kappa 7S34 and kappa 22S25 as second group offers the possibility of estimating the minimal number of kappa germline gene involved in the immune response to the structurally defined streptococcal group polysaccharide antigen ABSTRACT TRUNCATED AT WORDS

the anti phosphocholine PC memory response of BALB mice to PC KLH contains two groups of antibodies distinguished by fine specificity and by expression of the T15 idiotype that dominates group but not group II anti PC antibodies the contribution of gene to this diversity was investigated by the analysis of chains from PC binding hybridoma protein PCBHP representative of group and group II terminal amino-acid sequence analysis was performed on the chains of three independently derived group II PCBHP up to residue PCG1 or aPC and aPC these three sequence differed from each other by only one or two residue but differed by approximately from the chains of the group like PC binding myeloma protein PCBMP the group II sequence are closely related to isoelectric focusing analysis was also performed on the chain of PCG1 as well as on chains from PCBHP typical of group antibodies and from an atypical PCBHP differing from groups and II in fine specificity group PCBHP and the atypical PCBHP expressed chains related to and respectively the chains of another group PCBHP and of the group II protein PCG1 appeared different from those found in the PCBMP and from each other the results indicate more diverse expression of chains in the memory anti PC response than is represented by the PCBMP both and derived chains and presumably somatic variants as well as products of additional gene appear to be present in the anti PC memory pool

proteins and polypeptides are derivatized with dimethylaminoazobenzene isothiocyanate DABITC before their separation on sodium dodecyl sulfate polyacrylamide gels DABITC derivatized protein are detected visually in the picomole range without further staining and destaining procedures the recovered colored protein can also be used for direct sequence determinations this method was applied to purify heavy and light chains of murine hybridoma derived antibodies starting with ammonium sulfate precipitated protein mixture this method allows in one step procedure the isolation of pure light and heavy chains suitable for automatic sequencing

anti hapten antibodies to phosphorylcholine PC conjugated protein can be divided into antibodies reacting preferentially with PC group or with PC phenyl group II We have selected two hybridomas producing group II type anti PC antibodies of the IgM and IgE classes respectively with one exception NH2 terminal amino-acid sequence analyses of the light chains are identical in their first residue and similar to that of M460 but differ from that of group type light chains these results suggest that PC phenyl specific group II antibodies are expressed independently of PC specific group antibodies

the first complete sequence of the variable region of kappa light chain kappa from mouse anti streptococcal group polysaccharide antibody immunoglobulin 7S34 is reported immunoglobulin 7S34 was isolated from the ascitic fluid of hybridoma 7S34 previously cloned in vitro newly developed technique for the isolation of peptide by using pre column formation of peptide derivatives with dimethylaminoazobenzene isothiocyanate also served to complete the sequence the sequence of the variable region of the kappa light chain of immunoglobulin 7S34 defines new mouse kappa isotype kappa and is the first mouse immunoglobulin light chain variable region to be shown to have an extra cysteine residue at position

antibodies 2S1 and 2S1 are hybridoma derived gamma 3K murine immunoglobulins with specificity for acetylglucosamine beta leads to linked to rhamnose the immunodeterminant of the streptococcal group polysaccharide the VL 2S1 amino-acid sequence is the second complete one reported with this specificity and it is the first fully determined VK25 structure the VK regions of this and the previously described monoclonal anti CHO antibody 7S34 are compared with selected partial and complete murine VK regions preferentially from anti polysaccharide antibodies both VK 2S1 and 7S34 increase the variability of existing murine VK regions however they are as anti CHO antibodies most homologous to each other sharing the so far unique residue argine the amino-acid sequence of the constant region of 2S1 light chain was also determined It was found to be identical with that of MOPC21 but different from MOPC41 and MOPC70 in residue positions and

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