ITRAQ实验原理.ppt

ITRAQ实验原理简介,Section1:实验原理,定量蛋白质组学技术,SILACICAT/iTRAQ/O18,IsotopeTaggingforRelativeandAbsoluteproteinQuantitation(iTRAQ),CandeferentiallylabelandrunuptoEightsamplesProteinsaredigestedpriortolabelingLabelsreactwithN-terminusNoreductionofpeptidesbasedonaminoacidcompositionAnalyzeallpeptidesMassofpeptideionsisthesameallowingforasinglemasstobeselectedforMS/MSReportergroupislostduringfragmentationUsedtodeterminerelativeabundanceofselectedpeptideofinterestfromthefour(oreight)samples,iTRAQreagentstructure,iTRAQLabeling,iTRAQPrinciple,Ross,P.L.etal.(2004)MolCellProteomics3(12):1154-69,iTRAQ,Sample1,Sample2,Sample3,Sample4,Reduceeightpresentedasosteoarthritis(OA)patientsandsevenwereagematchedcontrols.OApatientsweredefinedashavingfrequentkneesymptomsinthepastyear,aKellgrenandLawrencegrades2or3ononeorbothknees,andabodymassindex(BMI)g30.Age-matchedcontrolvolunteershadnoevidenceofOAinthekneeandaBMI25)inthesurveyscansequentiallysubjectedtoproductionanalysis.4.Productionspectrawereaccumulatedfor2sinthemassrangem/z100-2000withamodifiedEnhanceAllmodeQ2transitionsettingfavoringlowmassionssothatthereportingiTRAQion(114,115,116and117m/z)intensitieswereenhancedforquantitation.Dynamicexclusionwasusedwitha2minand150ppmwindow.,DataAnalysis-1(eg),PeptideselectioncriteriaforrelativequantitationReplicateanalysisandcutoffpointestimationSignificantdifferentiallyregulatedproteinProteinclassificationValidationofiTRAQidentifiedCandidates,合作客户网络图,项目合作客户分类,合作的科研院校近200家:包括复旦大学、北京大学、上海交大、华东师大、军科院、中科院生物物理所、第四军医大学、广州中山大学中科院北京植物所、昆明动物所、华中农业大学、香港浸会大学等；临床医院近150家:包括上海瑞金医院、上海中山医院、上海华山医院、上海第六人民医院、上海长海医院、上海长征医院、北京阜外医院、