reproducibility of an hplc-esi-msms method for the measurement of stable-isotope enrichment of in vivo-labeled muscle atp synthase beta subunit再现性hplc-esi-msms方法测量的稳定同位素浓缩的vivo-labeled肌肉atp合酶β亚基.pdfVIP

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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再现性hplc-esi-msms方法测量的稳定同位素浓缩的vivo-labeled肌肉atp合酶β亚基.pdf

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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再现性hplc-esi-msms方法测量的稳定同位素浓缩的vivo-labeled肌肉atp合酶β亚基

Reproducibility of an HPLC-ESI-MS/MS Method for the Measurement of Stable-Isotope Enrichment of in Vivo- Labeled Muscle ATP Synthase Beta Subunit Sarah Everman1,2, Zhengping Yi1,2,3, Paul Langlais1,2, Lawrence J. Mandarino1,2, Moulun Luo1,2, Christine Roberts1,2, Christos S. Katsanos1,2* 1 Center for Metabolic and Vascular Biology, School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America, 2 Mayo Clinic Arizona, Scottsdale, Arizona, United States of America, 3 Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy/Health Sciences, Wayne State University, Detroit, Michigan, United States of America 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 a rat infused with stable-isotope-labeled leucine. The muscle was homogenized, b-F1-ATPase immunoprecipitated, and the protein was resolved using 1D-SDS PAGE. Following trypsin digestion of the isolated protein, the resultant peptide mixtures were subjected to analysis by HPLC-ESI-MS/MS, which resulted in the detection of multiple b- F1-ATPase peptides. There were three b-F1-ATPase unique peptides with a leucine residue in the amino acid sequence, and which were detected with high intensity relative to other peptides and assigned with .95% probability to b-F1-ATPase. These peptides were specifically targeted for fragmentation to access their stable-isotope enrichment based on MS/MS peak areas calculated from extracted ion chromatographs for selected labeled and unlabeled fragment ions. Results showed best lin