蛋白质组学技术的详细讲座(非常详细)

QuantitativeProteomics:ApplicationsandStrategies,October2013,Alittlehistory,1985Firstuse:uptoa3kDapeptidecouldbeionized1987Methodtoionizeintactproteins(upto34kDa)describedInstrumentshavenosequencecapability1989ESIisusedforbiomolecules(peptides)Sequencecapability,butlowsensitivity1994TermProteomeiscoined1995LC-MS/MSisimplementedGoldstandardofproteomicanalysis,2DE-basedapproach,2DE-basedapproach,“Isee1000spots,butidentify50only.”,Fennetal.,Science246:64-71,1989.,LC-MS,MS-basedquantitation,Inlet,IonSource,MassAnalyzer,Detector,MALDIES,Time-of-FlightQuadrupoleIonTrapQuadrupole-TOF,LC,Peakintensitiescanvaryupto100 xbetweenduplicateruns.,QuatitativeanalysisMUSTbecarriedonasinglerun.,IonIntensity=Ionabundance,MSmeasurem/z,m/z,Intensity,Sample2,Sample1,IsotopicLabeling,Unlabeledpeptide:,Labeledpeptide:,a),EnzymaticLabeling,MetabolicLabeling,SILAC,X3,X3,Cellsinnormalculturemedia,OngSEetal.,2002,ChemicalLabeling,GygiSPetal.,1999,ICAT(Isotope-CodedAffinityTag),ICAT,Thiol-specificgroup=bindstoCysteins,ICAT,Thiol-specificgroup=bindstoCysteins,QuantitationatMS1level,m/z,Intensity,Doublesamplecomplexity,i.e.instrumenthavemore“features”toidentify,i.e.decreaseinidentificationrate,iTRAQ(isobaricTagforRelativeandAbsoluteQuantitation),Sampleprep,Totalmassoflabel=145DaALWAYS,RecognizesArgorLys,iTRAQ,iTRAQ,Multiplexing,MetabolicVSChemicalLabeling,Summary,KolkmanAetal.,2005,Label-free,Mobilephase,A,A=5%organicsolventinwaterB=95%organicsolventinwater,B,C18column,25cmlong,20s,Label-free,StrassbergerVetal.,2010,Summary,Summary,Takehomemessage,Quantitationcanbedonegel-freeLabelingcanbeperformedatproteinorpeptidelevel,duringnormalcellgrowthorinvitroQuantitationcanbeachievedatMS1orMS2levelMethodchoicedependsonexperimentaldesign,costs,expertiseetcInmyPERSONALOPINION,chemicallabelshouldbeavoidedatallcostsunlessheavymultiplexingisrequired,Applications,Upregulatedprotein-Peptideratio1,ControlvsTumorCell?Controlvsdrugtreatedcell?Controlvsknock-outcell?,ApplicationsCellBiology,GeigerTetal.,2012,ApplicationsCellBiology,ApplicationsImmunology,Meissneretal,Science2013,ClinicalProteomics,A.AmyloidtissuestainedinCongoRed;B.AfterLMD.,WisniewskiJRetal.,2012,Interactomics,SchulzeandMann,2004SchulzeWXetal.,2005,SignalingPathways,Takehomemessage,Anythingispossible!,SILAC,SILAC,X3,X3,Cellsinnormalculturemedia,OngSEetal.,2002,ImportanceofDialyzedSerum,non-dialzedserumcontainsfree(unlabeled)aminoacids!,Noalterationstocellphenotype,C2C12myoblastcellline,Labeledcellsbehavedasexpectedunderdifferentiationprotocols,WhySILACisconvenient?,WhySILACisconvenient?,Convenient-noextrastepintroducedtoexperiment,justspecialmediumLabelingisguaranteedcloseto99%.AllidentifiedproteinsinprinciplearequantifiableQuantitationofproteinsaffectedbydifferentstimuli,disruptionofgenes,etc.Quantitationofpost-translationalmodifications(phosphorylation,etc.)Identificationandquantitationofinteractionpartners,Catch22,SILACcustomformulationmedia(withoutLysand/orArg)$LabeledaminoacidsLys4,Lys6,Lys8,Arg6,Arg10.Useformulationaccordinglytomediaformula(RPMILys,40mg/L)*WhendoingArglabeling,attentiontoProlineconversion!(50%oftrypticpeptidesinarandommixturepredictedtocontain1Pro),ProlineConversion!,TypicalSILACexperimentworkflow,Upregulatedprotein-Peptideratio1,Backgroundprotein-Peptideratio1:1,Additionalvalidationcriteria,*NeveruselabelledArgorLyswithsamemassdifference(Lys6/Arg6),TripleSILAC,TripleEncodingSILACallows:MonitoringofthreecellularstatessimultaneouslyStudyofthedynamicsofsignaltransductioncascadeseveninshorttimescales,BlagoevBetal.,2004,Fivetime-point“multiplexing”profile,BlagoevBetal.,2004,QuantitativephosphoproteomicsinEGFRsignaling,BlagoevBetal.,2004,SILAC-HeLacells,0EGF,1EGF,5EGF,5EGF,10EGF,20EGF,0-5-10min.Cytoplasmicext.Nuclearextract,LysisandFractionationAnfdigestion,1-5-20min.Cytoplasmicext.Nuclearextract,SCX/TiO2,SCX/TiO2,SCX/TiO2,SCX/TiO2,Phospho-peptideenrichment,44LC-MSruns,4x(10SCX-frac-tions+FT),IDandquantitation,8x,8x,8x,8x,8x,8x,MAPkinasesactivation,Signalprogression,Spatialdistributionofphosphorylationdynamics,CytosolicSTAT5translocatestothenucleusuponphosphorylation,Interactomics,SchulzeandMann,2004SchulzeWXetal.,2005,Limitations,ExpensiveQuantitationatMS1levelincreasedsamplecomplexityCellshastogrowinculture.Notachoiceforprimarycells,tissuesorbodyfluids.Celllineshavetobedyalizedserum-friendly.,SILAC-labeledorganism,SuryMDetal.,2010,Super-SILAC,GeigerTetal.,2010,Spike-InSILAC,GeigerTetal.,2013,Takehomemessage,Arguablythebestlabelingstrategies:easytohandle,nochemicalsteps,98%incorporationlowvariabilitySuccessfullyusedinthemostdiverseapplicationsCellsmustbestableandgrowinginthemediaTherearedecentalternativestrategiesforprimarycellsororganisms.,Label-free,October2013,Label-free,Label-free,StrassbergerVetal.,2010,10s,500fmolpeptide,100fmolpeptide,Label-free,KiyonamiR.etal,Thermo-Finniganapplicationnote500,2010.,Label-free,Label-free,StrassbergerVetal.,2010,Label-free,Neilsonetal.,Proteomics2011,SpectralCount,899.013,899.013,899.013,MS1(orMS),MS2(orMS/MS),SpectralCount,20s,Dependingonhowcomplexthesampleisataspecificretentiontime,themachinemightbebusy(i.e.,doingmanyMS2)oridle(i.e.,fewornoneMS2),LimitationinSpectralCount,MSscan,MS2scan,2counts,2counts,AreaUnderCurvemeasurement,RetentionTime,IonIntensity,AreaUnderCurvemeasurement,MS2scan,IonintensityinoneMS1,RetentionTime,ImportanceofResolutionforlabel-free,2+,2+,CoxandMann,NatureBiotechnol26,2008.,Label-freebecamereliable(*),ImportanceofResolutionforlabel-free,1,2,3,x,CoxandMann,NatureBiotechnol26,2008.,AreaUnderCurvemeasurement,RegardingLabel-free,Calculateindividualpeptide“Intensity”.ProteinIntensity=meanofpeptidesintensitiesLFQnormalization,DatawithoutNormalization,7422proteinsidentified7105proteinsquantified(95.72%),Howthiswasdemonstrated?,Yeastmodel,GhaemmagamiS.etal.,Nature425,2003,HuhWK.etal.,Nature425,2003,Howthiswasdemonstrated?,GhaemmagamiS.etal.,Nature425,2003,MaxQuantandYeast,DeGodoyLM.etal,2008.,Label-freebecamereliableANDshowedgoodcorrelationwithawell-establishedmodel,Label-freeinprimarycells,HigherCD4+,HigherCD8+,PatternRecognitionReceptorsPathway,InfectionwithSendaivirus(activateRIG-IPRR),RIG-Iknockout,Label-freeinprimarycells,Takehomemessage,“Labe-free”representsamyriadofANYmethodthatdoesnotuseanylabelingAreaUnderCurvecalculationsarethemostappropriateReliabilityisheavilydependentingoodinstrumentationandgoodbioinformatics(MaxQuant)Currently,almostasgoodasSILAC(yetslightlylessaccurate),SRM/MRM,October2013,Alittlehistory,Sofar,IDeverythingwecan,Mobilephase,A,B,C18column,25cmlong,20s,Targetedanalysis,Insomecases,theresearcherdontwanttheMSinstrumenttowastetimetryingtosequenceasmuchaspossible,butjustto“search”andsequencepre-determinedpeptides.BiomarkerresearchTrackingspecificmetabolicpathwaysTrackinglowabundantproteinsinchallengingsample(f.ex.,inserum),Plasmadynamicrange,SchiessRetal.,2009,Improvingdetectionthroughtergeting,MichalskiAetal.,2011,Biomarker,Discoveryphase,Screeningthesamplegivesyouthefollowinginfo:ForproteinXmostintensepeptides(notallpeptidesfromsameproteinhavethesameintensity)-mostcommonm/zformat(+2,+3,PTM?)-theirRetentiontimes-theirfragmentationprofiles(doesthe+2fragmentswell?),Biomarker,Shortergradient=MorecomplexMS1,Asyoudecreaseseparationresolution,youincreasethechancethattwoormorepeptideswithdifferentsequencesBUTveryclosem/zelutesatthesametime.,SRM(SelectedReactionMonitoring),Differenttransitionsfromsamepeptide,Performancewithsyntheticpeptides,Shortergradient=MorecomplexMS1,Asyoudecreaseseparationresolution,youincreasethecha