Sequence-Specific Capture of Protein-DNA Complexes for Mass Spectrometric Protein Identification 英文参考文献.docVIP

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Sequence-Specific Capture of Protein-DNA Complexes for Mass Spectrometric Protein Identification 英文参考文献.doc

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Sequence-Specific Capture of Protein-DNA Complexes for Mass Spectrometric Protein Identification 英文参考文献

Sequence-SpecificCaptureofProtein-DNAComplexes forMassSpectrometricProteinIdentification Cheng-HsienWu1,2,SiyuanChen1,MichaelR.Shortreed1,GloriaM.Kreitinger1,YuanYuan1,BrianL. Frey1,YiZhang3,ShamaMirza3,4,LisaA.Cirillo5,MichaelOlivier3,6,LloydM.Smith1,2,7 * 1DepartmentofChemistry,UniversityofWisconsin,Madison,Wisconsin,UnitedStatesofAmerica,2PrograminCellularandMolecularBiology,UniversityofWisconsin- Madison,Madison,Wisconsin,UnitedStatesofAmerica,3BiotechnologyandBioengineeringCenter,MedicalCollegeofWisconsin,Milwaukee,Wisconsin,UnitedStatesof America,4DepartmentofBiochemistry,MedicalCollegeofWisconsin,Milwaukee,Wisconsin,UnitedStatesofAmerica,5DepartmentofCellBiology,Neurobiology,and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America, 6Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin,UnitedStatesofAmerica,7GenomeCenterofWisconsin,Madison,Wisconsin,UnitedStatesofAmerica Abstract Theregulationofgenetranscriptionisfundamentaltotheexistenceofcomplexmulticellularorganismssuchashumans. Althoughitiswidelyrecognizedthatmuchofgeneregulationiscontrolledbygene-specificprotein-DNAinteractions,there presentlyexistslittleinthewayoftoolstoidentifyproteinsthatinteractwiththegenomeatlocationsofinterest.Wehave developed a novel strategy to address this problem, which we refer to as GENECAPP, for Global ExoNuclease-based EnrichmentofChromatin-AssociatedProteinsforProteomics.Inthisapproach,formaldehydecross-linkingisemployedto covalentlylinkDNAtoitsassociatedproteins;subsequentfragmentationoftheDNA,followedbyexonucleasedigestion, produces a single-stranded region of the DNA that enables sequence-specific hybridization capture of the protein-DNA complexonasolidsupport.Massspectrometric(MS)analysisofthecapturedproteinsisthenusedfortheiridentification and/or quantification. We show here the development and optimization of GENECAPP for an in vitro model system, comprisedofthemurineinsulin-likegrowthfactor-bindingprotein1(IG