Reproducibility of an HPLC-ESI-MS/MS method for the measurement of stable-isotope enrichment of in vivo-labeled muscle ATP synthase beta subunit.

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 ? subunit (?-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, ?-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 ?-F1-ATPase peptides. There were three ?-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 ?-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 linearity (R(2)?=?0.99) in the detection of MS/MS peak areas for both labeled and unlabeled fragment ions, over a wide range of amounts of injected protein, specifically for the ?-F1-ATPase(134-143) peptide. Measured stable-isotope enrichment was highly reproducible for the ?-F1-ATPase(134-143) peptide (CV?=?2.9%). Further, using mixtures of synthetic labeled and unlabeled peptides we determined that there is an excellent linear relationship (R(2)?=?0.99) between measured and predicted enrichment for percent enrichments ranging between 0.009% and 8.185% for the ?-F1-ATPase(134-143) peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle ?-F1-ATPase based on the determination of the enrichment of the ?-F1-ATPase(134-143) peptide.

SUBMITTER: Everman S 

PROVIDER: S-EPMC3192170 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature