应用电子捕获解离质谱对蛋白质磷酸化位点的定位.ppt

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1、LargeScaleLocalizationofProteinPhosphorylationbyUseofElectronCaptureDissociationMassSpectrometrySteveM.M.Sweet‡§,ChristopherM.Bailey‡§,DebbieL.Cunningham‡§,JohnK.Heath‡§,andHelenJ.Cooper§¶Fromthe‡CancerResearchUKGrowthFactorGroup,§SchoolofBiosciences,CollegeofLifeandEnvironmenta

2、lSciences,UniversityofBirmingham,Edgbaston,BirminghamB152TT,UnitedKingdomContentIntroductionofbackgroundExperimentproceduresAnalysisofResultsConclusions1.IntroductionofbackgroundDamagetostructureIncontrasttotraditionmathods,labilemodificationsareusuallyretainedintactuponECDpeptide

3、backbonecleavage.LowprecisionLackofstudyECDprovidesmoreextensivesequencecoverageinpolypeptides，andathigherelectronenergiesevenisoleucineandleucinearedistinguishable．NolargescalecomparisonofCIDandECDforphosphoproteinanalysishasbeencarriedout.2.ExperimentproceduresCellCultureInvitro

4、genCelllysisCentrifugationDeterminedbyCoomassieAcidificationanddesaltedStrongCationExchange(SCX)ChromatographyPhosphopeptideEnrichmentLC-MS/MSData-dependentCIDandECD(DD-CID-ECD)DataAnalysis3.AnalysisofResultsSummaryofphosphopeptidesidentifiedfrombothDD-CID-ECDandNL-ECDexperiments.

5、Theresultsindicatethatphosphopeptidesconstituted40–45%ofthestartingmixture.ItshouldbenotedthatnoteveryphosphopeptideidentifiedbyCIDtriggeredanECDevent.FIG.1.OverlapbetweenDTAsleadingtoidentificationsbyECDandCID.All4763identifications(IDs)arefrompairedCID/ECDevents.FIG.2.Overlapbet

6、weendistinctphosphopeptidesidentifiedbyECDandCID.All1220identificationsarefrompairedCID/ECDevents.FIG.3.BinneddistributionofSLoMoscores.Thexaxishaslogscale.AllidentificationsarefrompairedCID/ECDevents.Identificationswithonlyonepossiblesiteoflocalizationarenotincluded.FIG.4.Overlap

7、betweendistinct,welllocalized(SLoMo>19)phosphopeptidesidentifiedbyECDandCID.All725identificationsarefrompairedCID/ECDevents.Identificationswithonlyonepossiblelocalizationarenotincluded.FIG.5.Exampleofisomericphosphopeptideco-elution.a,ECDmassspectrumshowingfirstandthirdserinephosp

8、horylation(SLoMoscore,21.5).b,CID