Reproducibility of an HPLC-ESI-MS/MS method for the measurement of stable-isotope enrichment of in vivo-labeled muscle ATP synthase beta subunit
| Autor: | Lawrence J. Mandarino, Paul R. Langlais, Zhengping Yi, Sarah Everman, Christine Roberts, Christos S. Katsanos, Moulun Luo |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
Male
Proteomics Anatomy and Physiology Mitochondrial Diseases lcsh:Medicine Peptide Biochemistry Rats Sprague-Dawley 0302 clinical medicine Isotopes lcsh:Science Peptide sequence Musculoskeletal System Chromatography High Pressure Liquid Protein Metabolism chemistry.chemical_classification 0303 health sciences Multidisciplinary Radiochemistry biology ATP synthase Chemistry Muscles Mitochondrial Proton-Translocating ATPases Isotope Labeling Muscle Medicine Leucine ATP synthase alpha/beta subunits Research Article Signal peptide Spectrometry Mass Electrospray Ionization Molecular Sequence Data 030209 endocrinology & metabolism Mass spectrometry 03 medical and health sciences In vivo Genetics Animals Amino Acid Sequence Biology 030304 developmental biology Clinical Genetics lcsh:R Reproducibility of Results Human Genetics Rats Metabolism biology.protein lcsh:Q Peptides |
| Zdroj: | PLoS ONE, Vol 6, Iss 10, p e26171 (2011) PLoS ONE |
| ISSN: | 1932-6203 |
| Popis: | 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 β subunit (β-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, β-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 β-F1-ATPase peptides. There were three β-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 β-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 β-F1-ATPase(134-143) peptide. Measured stable-isotope enrichment was highly reproducible for the β-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 β-F1-ATPase(134-143) peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle β-F1-ATPase based on the determination of the enrichment of the β-F1-ATPase(134-143) peptide. |
| Databáze: | OpenAIRE |
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