Journal
ANALYTICAL BIOCHEMISTRY
Volume 403, Issue 1-2, Pages 1-12Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2010.04.018
Keywords
Peptide liquid chromatography/mass spectrometry; Heavy water labeling; Major histocompatibility complex proteins; Allelic polymorphism; Mass isotopomer distribution analysis; Protein synthesis
Funding
- Cambridge Arthritis Research Endeavour
- Stanley Elmore Senior Research Fellowship at Sidney Sussex College
- Arthritis Research UK [18543]
- Versus Arthritis [18543] Funding Source: researchfish
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Methodological limitations have hampered the use of heavy water ((H2O)-H-2), a convenient, universal biosynthetic label, for measuring protein synthesis. Analyses of H-2-labeled amino acids are sensitive to contamination; labeling of peptides has been measured for a few serum proteins, but this approach awaits full validation. Here we describe a method for quantifying protein synthesis by peptide mass spectrometry (MS) after (H2O)-H-2 labeling, as applied to various proteins of the major histocompatibility complex (MHC). Human and murine antigen-presenting cells were cultured in medium containing 5% (H2O)-H-2; class I and class II MHC proteins were immunoprecipitated, bands were excised, and Ala-/Gly-rich, allele-specific tryptic peptides were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mass isotopomer distributions were quantified precisely by LC-MS and shifted markedly on (H2O)-H-2 labeling. Experimental data agreed closely with models obtained by mass isotopomer distribution analysis (MIDA) and were consistent with contributions from Ala, Gly, and other amino acids to labeling. Estimates of fractional protein synthesis from peptides of the same protein were precise and internally consistent. The method was capable of discriminating between MHC isotypes and alleles, applicable to primary cells, and readily extendable to other proteins. It simplifies measurements of protein synthesis, enabling novel applications in physiology, in genotype/phenotype interactions, and potentially in kinetic proteomics. (C) 2010 Elsevier Inc. All rights reserved.
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