应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座

LargeScaleLocalizationofProtein

PhosphorylationbyUseofElectronCaptureDissociationMassSpectrometry应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第1页SteveM.M.Sweet‡§,ChristopherM.Bailey‡§,DebbieL.Cunningham‡§,

JohnK.Heath‡§,andHelenJ.Cooper§¶Fromthe‡CancerResearchUKGrowthFactorGroup,§SchoolofBiosciences,CollegeofLifeandEnvironmentalSciences,UniversityofBirmingham,Edgbaston,BirminghamB152TT,UnitedKingdom应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第2页应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第3页ContentIntroductionofbackgroundExperimentproceduresAnalysisofResultsConclusions应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第4页1.IntroductionofbackgroundDamagetostructureIncontrasttotraditionmathods,labilemodificationsareusuallyretainedintactuponECDpeptidebackbonecleavage.LowprecisionLackofstudyECDprovidesmoreextensivesequencecoverageinpolypeptides，andathigherelectronenergiesevenisoleucineandleucinearedistinguishable．NolargescalecomparisonofCIDandECDforphosphoproteinanalysishasbeencarriedout.应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第5页应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第6页应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第7页应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第8页应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第9页2.ExperimentproceduresCellCultureInvitrogenCelllysisCentrifugationDeterminedbyCoomassieAcidificationanddesaltedStrongCationExchange(SCX)ChromatographyPhosphopeptideEnrichmentLC-MS/MSData-dependentCIDandECD(DD-CID-ECD)DataAnalysis应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第10页3.AnalysisofResultsSummaryofphosphopeptidesidentifiedfrombothDD-CID-ECDandNL-ECDexperiments.Theresultsindicatethatphosphopeptidesconstituted40–45%ofthestartingmixture.ItshouldbenotedthatnoteveryphosphopeptideidentifiedbyCIDtriggeredanECDevent.应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第11页FIG.1.OverlapbetweenDTAsleadingtoidentificationsbyECDandCID.All4763identifications(IDs)arefrompairedCID/ECDevents.FIG.2.OverlapbetweendistinctphosphopeptidesidentifiedbyECDandCID.All1220identificationsarefrompairedCID/ECDevents.应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第12页FIG.3.BinneddistributionofSLoMoscores.Thexaxishaslogscale.AllidentificationsarefrompairedCID/ECDevents.Identificationswithonlyonepossiblesiteoflocalizationarenotincluded.应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第13页FIG.4.Overlapbetweendistinct,welllocalized(SLoMo>19)phosphopeptidesidentifiedbyECDandCID.All725identificationsarefrompairedCID/ECDevents.Identificationswithonlyonepossiblelocalizationarenotincluded.应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第14页应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第15页FIG.5.Exampleofisomericphosphopeptideco-elution.a,ECDmassspectrumshowingfirstandthirdserinephosphorylation(SLoMoscore,21.5).b,CIDmassspectrumshowingevidenceforthreoninephosphorylation(SLoMoscore,24.3).ThesiteofphosphorylationindicatedbySLoMoisshownuppermostinsetinb.应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第16页FIG.6.MultiplyphosphorylatedpeptidesidentifiedandlocalizedfromECDandCIDmassspectraandsortedbychargestate.Allidentificationsarefrompairedevents.Identificationswithonlyonepossiblelocalizationarenotincluded.应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第17页4.Conclusion

Inthisstudyweanalyzed6080pairedCID/ECDmassspectra,1892ofwhichledtophosphopeptideidentificationsbyCID,ECD,orboth.ThisdatasetallowedacomparisonoftherelativemeritsofLTQCIDandFT-ICRECDmassspectraforphosphopeptideidentificationandsitelocalization.应用电子捕获解离质谱对蛋白质磷酸化位点的定位专家讲座第18页Inconclusion,ourresultsindicatethatcombinedECDandCIDanalysisresultsinhighconfidencephosphopeptideidentificationsandphosphorylationsitelocalization.Hybridmassspectrometers,suchastheLTQ-FTusedinthiswork,areprimarilyusedtomeasureprecursorionswithhighmassaccuracywhilecarryingoutrapidCID(atlowerresolution).Weshowedthattherearepotentialadvantagestousingbothpar