Reproducibility of an HPLC-ESI-MSMS Method for the Measurement of Stable-Isotope Enrichment of in Vivo-Labeled Muscle ATP Synthase Beta Subunit 英文参考文献.docVIP

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Reproducibility of an HPLC-ESI-MSMS Method for the Measurement of Stable-Isotope Enrichment of in Vivo-Labeled Muscle ATP Synthase Beta Subunit 英文参考文献.doc

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Reproducibility of an HPLC-ESI-MSMS Method for the Measurement of Stable-Isotope Enrichment of in Vivo-Labeled Muscle ATP Synthase Beta Subunit 英文参考文献

ReproducibilityofanHPLC-ESI-MS/MSMethodforthe MeasurementofStable-IsotopeEnrichmentofinVivo- LabeledMuscleATPSynthaseBetaSubunit SarahEverman1,2,ZhengpingYi1,2,3,PaulLanglais1,2,LawrenceJ.Mandarino1,2,MoulunLuo1,2 ,Christine Roberts1,2,ChristosS.Katsanos1,2 * 1CenterforMetabolicandVascularBiology,SchoolofLifeSciences,ArizonaStateUniversity,Tempe,Arizona,UnitedStatesofAmerica,2MayoClinicArizona,Scottsdale, Arizona,UnitedStatesofAmerica,3DepartmentofPharmaceuticalSciences,EugeneApplebaumCollegeofPharmacy/HealthSciences,WayneStateUniversity,Detroit, Michigan,UnitedStatesofAmerica Abstract We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase b subunit (b-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is reduced under a variety of circumstances. Muscle was obtained from arat infused with stable-isotope-labeled leucine. Themuscle washomogenized, b-F1-ATPase immunoprecipitated,andtheproteinwasresolvedusing1D-SDSPAGE.Followingtrypsindigestionoftheisolatedprotein, theresultantpeptidemixturesweresubjectedtoanalysisbyHPLC-ESI-MS/MS,whichresultedinthedetectionofmultipleb- F1-ATPasepeptides.Therewerethreeb-F1-ATPaseuniquepeptideswithaleucineresidueintheaminoacidsequence,and whichweredetectedwithhighintensityrelativetootherpeptidesandassignedwith.95%probabilitytob-F1-ATPase. Thesepeptideswerespecificallytargetedforfragmentationtoaccesstheirstable-isotopeenrichmentbasedonMS/MSpeak areascalculatedfromextractedionchromatographsforselectedlabeledandunlabeledfragmentions.Resultsshowedbest linearity(R2=0.99)inthedetectionofMS/MSpeakareasforbothlabeledandunlabeledfragmentions,overawiderangeof amountsofinjectedprotein,specificallyfortheb-F1-ATPase134-143peptide.Measuredstable-isotopeenrichmentwashighly reproducible for the b-F1-ATPase134-143 peptide (CV=2.9%). Further, using mixtures of synthetic labeled