蛋白质组学技术的详细讲座(非常详细)

QuantitativeProteomics:ApplicationsandStrategiesOctober2013Alittlehistory…1985–Firstuse:uptoa3kDapeptidecouldbeionized1987–Methodtoionizeintactproteins(upto34kDa)describedInstrumentshavenosequencecapability1989–ESIisusedforbiomolecules(peptides)Sequencecapability,butlowsensitivity1994–Term«Proteome»iscoined1995–LC-MS/MSisimplemented«Goldstandard»ofproteomicanalysis2DE-basedapproach2DE-basedapproach“Isee1000spots,butidentify50only.”Gradientelution:200nl/minColumn(75mm)/spraytip(8mm)Reverse-phaseC18beads,3mmPlatin-wire2.0kVSampleLoading:500nl/minNoprecolumnorsplitESI15cmFennetal.,Science246:64-71,1989.LC-MSMS-basedquantitationInletIonSourceMassAnalyzerDetectorMALDIESTime-of-FlightQuadrupoleIonTrapQuadrupole-TOFLCPeakintensitiescanvaryupto100xbetweenduplicateruns.QuatitativeanalysisMUSTbecarriedonasinglerun.IonIntensity=IonabundanceMSmeasurem/zm/zIntensitySample2Sample1IsotopicLabelingUnlabeledpeptide:Labeledpeptide:a)b)a)b)18O16O15N14N13C12C2H1HStableIsotopeElement18O16O15N14N13C12C2H1HStableIsotopeElementEnzymaticLabelingMetabolicLabelingSILAC*m/zm/zPassagecellstoallowincorporationoflabelledAABy5celldoublingscellshaveincorporated*m/zm/zGrowSILAClabelledcellstodesirednumberofcellsforexperiment*m/zm/zStartSILAClabellingbygrowingcellsinlabellingmedia(labelledAA/dializedserum)m/zm/zMediawithNormalAA()MediawithLabelledAA(*)X3X3CellsinnormalculturemediaOngSEetal.,2002ChemicalLabelingBiotinBiotintagtagLinkerLinker(heavyorlight)(heavyorlight)ThiolspecificThiolspecificreactivegroupreactivegroupICATReagents:ICATReagents:Heavyreagent:d8Heavyreagent:d8--ICAT(ICAT(XX=deuterium)=deuterium)Lightreagent:d0Lightreagent:d0--ICAT(ICAT(XX=hydrogen)=hydrogen)SNNONOOONIOOXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXGygiSPetal.,1999ICAT(Isotope-CodedAffinityTag)ICATCellState1CellState1((AllcysteineslabeledwithAllcysteineslabeledwithlightICATlightICAT))CellState2CellState2(Allcysteineslabeled(AllcysteineslabeledwithheavyICAT)withheavyICAT)RelativeAbundanceRelativeAbundance00100100ProteinAProteinAProteinBProteinBProteinCProteinCProteinDProteinDProteinEProteinEProteinFProteinF....200200400400600600800800200200400400600600800800m/zm/z00100100NHNH22--EACDPLREACDPLR--COOHCOOH=ProteinA=ProteinARelativeAbundanceRelativeAbundanceTimeTimeQuantitaterelativeproteinlevelsbymeasuringpeakratiosIdentifyproteinsbysequenceinformation(MS/MSscan)CombineCombineOptionalfractionationOptionalfractionationAffinityseparationAffinityseparationAnalyzebyLCAnalyzebyLC--MS/MSMS/MSProteolyzeProteolyzeThiol-specificgroup=bindstoCysteinsICATThiol-specificgroup=bindstoCysteinsQuantitationatMS1levelm/zIntensityDoublesamplecomplexity,i.e.instrumenthavemore“features”toidentify,i.e.decreaseinidentificationrateiTRAQ(isobaricTagforRelativeandAbsoluteQuantitation)SampleprepTotalmassoflabel=145DaALWAYSRecognizesArgorLysiTRAQiTRAQMultiplexingMetabolicVSChemicalLabeling•Metaboliclabeling-15Nlabeling-SILACLivingcellsEfficientlabelingSimple!•Chemicalmethods-many…butICATisprototypeIsolatedproteinsampleDependsonchemistryMulti-stepprotocolsRequireoptimizationSummaryKolkmanAetal.,2005Label-freeMobilephaseAA=5%organicsolventinwaterB=95%organicsolventinwaterBC18column,25cmlongTime20sLabel-freeStrassbergerVetal.,2010SummarySummaryTakehomemessage1.Quantitationcanbedonegel-free2.Labelingcanbeperformedatproteinorpeptidelevel,duringnormalcellgrowthorinvitro3.QuantitationcanbeachievedatMS1orMS2level4.Methodchoicedependsonexperimentaldesign,costs,expertiseetc5.InmyPERSONALOPINION,chemicallabelshouldbeavoidedatallcostsunlessheavymultiplexingisrequiredApplicationsStateAStateBLightIsotopeHeavyIsotopeMix1:1OptionalProteinFractionationDigestwithTrypsinProteinIdentificationandQuantitationbyLC-MSUpregulatedprotein-Peptideratio1Arg-12C6Arg-13C6m/zArg-12C6Arg-13C6m/zControlvsTumorCell?Controlvsdrugtreatedcell?Controlvsknock-outcell?Applications–CellBiologyGeigerTetal.,2012Applications–CellBiologyApplications–ImmunologyMeissneretal,Science2013ClinicalProteomicsA.AmyloidtissuestainedinCongoRed;B.AfterLMD.WisniewskiJRetal.,2012InteractomicsSchulzeandMann,2004SchulzeWXetal.,2005SignalingPathwaysTakehomemessage1.Anythingispossible!SILACSILAC*m/zm/zPassagecellstoallowincorporationoflabelledAABy5celldoublingscellshaveincorporated*m/zm/zGrowSILAClabelledcellstodesirednumberofcellsforexperiment*m/zm/zStartSILAClabellingbygrowingcellsinlabellingmedia(labelledAA/dializedserum)m/zm/zMediawithNormalAA()MediawithLabelledAA(*)X3X3CellsinnormalculturemediaOngSEetal.,2002ImportanceofDialyzedSerum•non-dialzedserumcontainsfree(unlabeled)aminoacids!NoalterationstocellphenotypeC2C12myoblastcelllineLabeledcellsbehavedasexpectedunderdifferentiationprotocolsWhySILACisconvenient?WhySILACisconvenient?•Convenient-noextrastepintroducedtoexperiment,justspecialmedium•Labelingisguaranteedcloseto99%.Allidentifiedproteinsinprinciplearequantifiable•Quantitationofproteinsaffectedbydifferentstimuli,disruptionofgenes,etc.•Quantitationofpost-translationalmodifications(phosphorylation,etc.)•IdentificationandquantitationofinteractionpartnersCatch2