蛋白质组学技术的详细讲座(非常详细).ppt

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1、QuantitativeProteomics:ApplicationsandStrategiesOctober2013Alittlehistory…1985–Firstuse:uptoa3kDapeptidecouldbeionized1987–Methodtoionizeintactproteins(upto34kDa)describedInstrumentshavenosequencecapability1989–ESIisusedforbiomolecules(peptides)Sequencecapability,

2、butlowsensitivity1994–Term«Proteome»iscoined1995–LC-MS/MSisimplemented«Goldstandard»ofproteomicanalysis2DE-basedapproach2DE-basedapproach“Isee1000spots,butidentify50only.”Gradientelution:200nl/minColumn(75mm)/spraytip(8mm)Reverse-phaseC18beads,3mmPlatin-wire2.0kVS

3、ampleLoading:500nl/minNoprecolumnorsplitESI15cmFennetal.,Science246:64-71,1989.LC-MSMS-basedquantitationInletIonSourceMassAnalyzerDetectorMALDIESTime-of-FlightQuadrupoleIonTrapQuadrupole-TOFLCPeakintensitiescanvaryupto100xbetweenduplicateruns.QuatitativeanalysisMU

4、STbecarriedonasinglerun.IonIntensity=IonabundanceMSmeasurem/zm/zIntensitySample2Sample1IsotopicLabelingUnlabeledpeptide:Labeledpeptide:a)b)a)b)EnzymaticLabelingMetabolicLabelingSILAC*m/zm/zPassagecellstoallowincorporationoflabelledAABy5celldoublingscellshaveincorp

5、orated*m/zm/zGrowSILAClabelledcellstodesirednumberofcellsforexperiment*m/zm/zStartSILAClabellingbygrowingcellsinlabellingmedia(labelledAA/dializedserum)m/zm/zMediawithNormalAA()MediawithLabelledAA(*)X3X3CellsinnormalculturemediaOngSEetal.,2002ChemicalLabelingGygi

6、SPetal.,1999ICAT(Isotope-CodedAffinityTag)ICATThiol-specificgroup=bindstoCysteinsICATThiol-specificgroup=bindstoCysteinsQuantitationatMS1levelm/zIntensityDoublesamplecomplexity,i.e.instrumenthavemore“features”toidentify,i.e.decreaseinidentificationrateiTRAQ(isoba

7、ricTagforRelativeandAbsoluteQuantitation)SampleprepTotalmassoflabel=145DaALWAYSRecognizesArgorLysiTRAQiTRAQMultiplexingMetabolicVSChemicalLabelingSummaryKolkmanAetal.,2005Label-freeMobilephaseAA=5%organicsolventinwaterB=95%organicsolventinwaterBC18column,25cmlon

8、gTime20sLabel-freeStrassbergerVetal.,2010SummarySummaryTakehomemessageQuantitationcanbedonegel-freeLabelingcanbeperformedatproteinorpeptidelevel,during