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 Everman, Zhengping Yi, Paul Langlais, Lawrence J. Mandarino, Moulun Luo, Christine Roberts, Christos S. Katsanos

Research output: Contribution to journalArticle

2 Citations (Scopus)

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 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.

Original languageEnglish (US)
Article numbere26171
JournalPloS one
Volume6
Issue number10
DOIs
StatePublished - Oct 12 2011
Externally publishedYes

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H-transporting ATP synthase
Proton-Translocating ATPases
Isotopes
reproducibility
stable isotopes
Muscle
high performance liquid chromatography
Adenosine Triphosphate
High Pressure Liquid Chromatography
peptides
Muscles
muscles
Peptides
methodology
Ions
Leucine
ions
leucine
Proteins
electrospray ionization mass spectrometry

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Reproducibility of an HPLC-ESI-MS/MS method for the measurement of stable-isotope enrichment of in vivo-labeled muscle ATP synthase beta subunit. / Everman, Sarah; Yi, Zhengping; Langlais, Paul; Mandarino, Lawrence J.; Luo, Moulun; Roberts, Christine; Katsanos, Christos S.

In: PloS one, Vol. 6, No. 10, e26171, 12.10.2011.

Research output: Contribution to journalArticle

Everman, Sarah ; Yi, Zhengping ; Langlais, Paul ; Mandarino, Lawrence J. ; Luo, Moulun ; Roberts, Christine ; Katsanos, Christos S. / Reproducibility of an HPLC-ESI-MS/MS method for the measurement of stable-isotope enrichment of in vivo-labeled muscle ATP synthase beta subunit. In: PloS one. 2011 ; Vol. 6, No. 10.
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