《蛋白质组学》PPT课件

系统生物学与蛋白质组学Introductiontosystematicbiology2：蛋白质经过还原，封闭后胰蛋白酶酶切;3：肽段混合物分别用不同的iTRAQ标记;4：等量混合各种iTRAQ试剂标记的肽段;5：MS/MS质谱检测及分析。,4:SILAC,SILAC法。细胞培养条件下稳定同位素标记技术(Stableisotopelabelingwithaminoacidsincellculture，SILAC)1、标记：在缺乏Lys和Arg的培养基中添加Lys和Arg的同位素Lys(D4或13C615N4)、Arg(13C6,或13C615N4)等培养细胞，使蛋白质被标记成“中型”或“重型”；2、细胞处理，如药物处理；3、细胞裂解、提取蛋白质；4、等量混合对照与处理组蛋白质、SDS-PAGE电泳、染色；5、割取蛋白质条带，胰蛋白酶消化，质谱分析。,MassSpectrometry:Methods&Theory,MassSpectrometry,Analyticalmethodtomeasurethemolecularoratomicweightofsamples,PeptideMassFingerprinting(PMF),MassSpecPrinciples,Ionizer,Sample,+,_,MassAnalyzer,Detector,m/zratio:,Molecularweightdividedbythechargeonthisprotein,Allproteinsaresortedbasedonamasstochargeratio(m/z),TypicalMassSpectrum,aspirin,RelativeAbundance,120m/z-forsinglychargedionthisisthemass,ResolutioninMS,ResolutioninMS,QTOF,783.455,784.465,785.475,783.6,SoftIonizationMethods,337nmUVlaser,MALDI,cyano-hydroxycinnamicacid,Goldtipneedle,Fluid(nosalt),ESI,+,_,Query(MALDI)Spectrum,5001000150020002500,698,2098,1199,1007,609,450,2211(trp),1940(trp),Queryvs.Database,QueryMassesDatabaseMassListResults,450.2017(P21234)609.2667(P12345)664.3300(P89212)1007.4251(P12345)1114.4416(P89212)1183.5266(P12345)1300.5116(P21234)1407.6462(P21234)1526.6211(P89212)1593.7101(P89212)1740.7501(P21234)2098.8909(P12345),450.2201609.3667698.31001007.53911199.49162098.9909,2Unknownmasses1hitonP212343hitsonP12345ConcludethequeryproteinisP12345,Databasesearch,theoretical,experimental,Mas