Improving the depth of coverage in membrane proteomic studies through the use of lipid-based protein immobilization technology in parallel with methanol-facilitated solubilisation

[摘要] Lipid-basedproteinimmobilization(LPI)technologyisaplatformrecentlydevelopedtofacilitateshotgunmembraneproteomicstudiesbasedonananotechnologyframework.Proteoliposomesaregeneratedfromamembraneproteinpreparation.TheseproteoliposomesareimmobilizedontoanLPIchipandthensubjectedtoproteolysis.TheproteolyticpeptidesarethensubjectedtoLC/MSanalysisafterfractionationbySCXchromatography.Thefocusofthisstudywastoevaluatehowthedepthofcoverageofthemembraneproteomeofaparticularcelltypevariedasafunctionofthesamplepreparationmethodused.Humandermalfibroblasts(hDFs)andhumanbonemarrowmesenchymalstemcells(BM-hMSCs)weresubjectedtomembraneproteomicstudiesusingtwodifferentsamplepreparationmethods:LPItechnologyandmethanol-facilitatedsolubilisation.ThenumberofmembraneproteinsthatcouldbeidentifiedfromhDFsandBM-hMSCsusingLC/MSwasgreaterusingLPItechnologythanitwasusingmethanol-facilitatedsolubilisation.However,thenumberofmembraneproteinidentificationsthatcouldbemadeforbothhDFsandBM-hMSCsincreasedby∼50%whenbothsamplepreparationmethodswereusedinparallelandtheMS/MSdatawasconvolvedtogether.Therefore,LPItechnologyisaveryusefultechnologyforhigh-throughputshotgunmembraneproteomicstudies.However,inordertomaximizethedepthofmembraneproteomecoveragethatcanbeattainedforagivencelltype,itisnecessarytousemultiplesamplepreparationmethodsinconcert...

[学科分类] 分析化学