应用电子捕获解离质谱对蛋白质磷酸化位点的定位

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

2、yofBirmingham,Edgbaston,BirminghamB152TT,UnitedKingdomContentIntroductionofbackgroundExperimentproceduresAnalysisofResultsConclusions1.IntroductionofbackgroundDamagetostructureIncontrasttotraditionmathods,labilemodificationsareusuallyretainedintactuponECDpeptidebackbonecleavage.LowprecisionLackofstu

3、dyECDprovidesmoreextensivesequencecoverageinpolypeptides，andathigherelectronenergiesevenisoleucineandleucinearedistinguishable．NolargescalecomparisonofCIDandECDforphosphoproteinanalysishasbeencarriedout.2.ExperimentproceduresCellCultureInvitrogenCelllysisCentrifugationDeterminedbyCoomassieAcidificat

4、ionanddesaltedStrongCationExchange(SCX)ChromatographyPhosphopeptideEnrichmentLC-MS/MSData-dependentCIDandECD(DD-CID-ECD)DataAnalysis3.AnalysisofResultsSummaryofphosphopeptidesidentifiedfrombothDD-CID-ECDandNL-ECDexperiments.Theresultsindicatethatphosphopeptidesconstituted40–45%ofthestartingmixture.I

5、tshouldbenotedthatnoteveryphosphopeptideidentifiedbyCIDtriggeredanECDevent.FIG.1.OverlapbetweenDTAsleadingtoidentificationsbyECDandCID.All4763identifications(IDs)arefrompairedCID/ECDevents.FIG.2.OverlapbetweendistinctphosphopeptidesidentifiedbyECDandCID.All1220identificationsarefrompairedCID/ECDeven

6、ts.FIG.3.BinneddistributionofSLoMoscores.Thexaxishaslogscale.AllidentificationsarefrompairedCID/ECDevents.Identificationswithonlyonepossiblesiteoflocalizationarenotincluded.FIG.4.Overlapbetweendistinct,welllocalized(SLoMo>19)phosphopeptidesidentifiedbyECDandCID.All725identificationsarefrompairedCID/

7、ECDevents.Identificationswithonlyonepossiblelocalizationarenotincluded.FIG.5.Exampleofisomericphosphopeptideco-elution.a,ECDmassspectrumshowingfirstandthirdserinephosphorylation(SLoMoscore,21.5).b,CID