Perfluorooctanoic Acid for Shotgun Proteomics 英文参考文献.docVIP

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Perfluorooctanoic Acid for Shotgun Proteomics 英文参考文献.doc

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Perfluorooctanoic Acid for Shotgun Proteomics 英文参考文献

PerfluorooctanoicAcidforShotgunProteomics ChandraSekharRaoKadiyala1,SaraE.Tomechko1,MasaruMiyagi1,2,3* 1Case Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio, United States of America, 2Department of Pharmacology, Case WesternReserveUniversity,Cleveland,Ohio,UnitedStatesofAmerica,3DepartmentofOphthalmologyandVisualSciences,CaseWesternReserveUniversity,Cleveland, Ohio,UnitedStatesofAmerica Abstract Here,wedescribethenoveluseofavolatilesurfactant,perfluorooctanoicacid(PFOA),forshotgunproteomics.PFOAwas foundtosolubilizemembraneproteinsaseffectivelyassodiumdodecylsulfate(SDS).PFOAconcentrationsupto0.5%(w/v) did not significantly inhibit trypsin activity. The unique features of PFOA allowed us to develop a single-tube shotgun proteomicsmethodthatusedallvolatilechemicalsthatcouldeasilyberemovedbyevaporationpriortomassspectrometry analysis.Theexperimentalproceduresinvolved:1)extractionofproteinsin2%PFOA;2)reductionofcystineresidueswith triethylphosphineandtheirS-alkylationwithiodoethanol;3)trypsindigestionofproteinsin0.5%PFOA;4)removalofPFOA by evaporation; and 5) LC-MS/MS analysis of the resulting peptides. The general applicability of the method was demonstratedwiththemembranepreparationofphotoreceptoroutersegments.Weidentified75proteinsfrom1m gof thetrypticpeptidesinasingle,1-hour,LC-MS/MSrun.About67%oftheproteinsidentifiedwereclassifiedasmembrane proteins.Wealsodemonstratethataproteolytic 18OlabelingprocedurecanbeincorporatedafterthePFOAremovalstep forquantitativeproteomicexperiments.Thepresentmethoddoesnotrequiresampleclean-updevicessuchassolid-phase extractionsandmembranefilters,sonoproteins/peptidesarelostinanyexperimentalsteps.Thus,thissingle-tubeshotgun proteomicsmethodovercomesthemajordrawbacksofsurfactantuseinproteomicexperiments. Citation: Kadiyala CSR, Tomechko SE, Miyagi M (2010) Perfluorooctanoic Acid for Shotgun Proteomics. PLoS ONE 5(12): e15332. doi:10.1371/ journal.pone.0015332 Editor:JeanKanellopoulos