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ID:51447348
大小:1.36 MB
页数:6页
时间:2020-03-24
《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
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