基于iTRAQ技术的水牛卵泡液蛋白质样品预处理的优化.pdf

《基于iTRAQ技术的水牛卵泡液蛋白质样品预处理的优化.pdf》由会员上传分享，免费在线阅读，更多相关内容在行业资料-天天文库。

1、南方农业学报JournalofSouthernA酣culture2014，45(11)：2046—2051ISSN2095-1191；CODENNNXAABhttp：／／www．nfnyxb．anDOI：10．3969／j：issn．2095-1191．2014．11．2006OptimizationofproteinsamplepreparationforbuffalofollicularfluidanalyzedwithiTRAQtechniqueHUWeil一，ZHOUJiel一，FUQian91'2+(1StateKeyLaboratoryforConservationandUt

2、ilizationofSubtropicalAgro-Bioresourees,Nanning2GuangxiUniversity，Nanning530005，China)Abstract：【Objective】Theoptimizationofproteinsamplepreparationextractedfrombuffalofollicularfluidwasconductedtostudydifferentialexpressionproteinbyusingisobarictagsforrelativeandabsolutequantification(i-TRAQ)tech

3、niqueinordertolaythefoumdationforiTRAQapplicationonproteindifferentialexpressionanalysis．【Method】Twodifferentprecipitantswereusedforprecipitatingproteinintheprocessofproteinsolutionenzymolysistocomparetheireffectsonproteinenzymolysis，iTRAQlabelingreactionandchromatographicseparationofpeptides．The

4、peptidessolutionwastreatedwitllspeedcolumnbefore。separatedbynanoliquidchromatography(nano—LC)toobserveitschromatographseparationeffectinordertoconfirmtheoptimalprocedureofproteinsamplepretreatment．【Result】Ascomparedtouse100％acetoneastheprecipitant，theproteinsolutionprecipitatedwith1：1volumeratioo

5、facetoneandethanolmixtureadding0．1％aceticacidcouldobtainidealprecipitationeffectsbeingwhiteprecipita-tionand5．76txg／lxLofproteincontent．Comparedwithnoprecipitationofproteinsolutionafterreductionandalky·lationintheprocessofenzymolysis，thehigherenzymolysisefficiency，andmuchpeptidepeakswithhigherint

6、ensitycouldbefoundinseparationchromatogramwhentheproteinsolutionwasprecipitatedwithacetoneandethanolmix—turebeforetrypsinenzymolysis．Asaresult，thelabelingreactionbetweeniTRAQreagentandpeptidewasveryeffec·tivetogethigherfragmentingefficiencyofprecursorionandmuchfragmentalpeaks，andtheiTRAQcharacter

7、isticreporterionof116m／zshowedbetterspectrogrampeakintensityandhigherresolutionthanthatof117m／z．Fur-thermore，theliquidchromatogramofpeptidesfromprecipitatedproteinsolutionpresentedmuchpeakswithhigherin-tensityanddistin