title: Measurement of protein synthesis using heavy water labeling and peptide mass spectrometry: Discrimination between major histocompatibility complex allotypes.
abstract: Methodological limitations have hampered the use of heavy water ((2)H(2)O), a convenient, universal biosynthetic label, for measuring protein synthesis. Analyses of (2)H-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 (2)H(2)O labeling, as applied to various proteins of the major histocompatibility complex (MHC). Human and murine antigen-presenting cells were cultured in medium containing 5% (2)H(2)O; 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 (2)H(2)O 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.
authors.affiliation: Department of Medicine, University of Cambridge, Cambridge CB2 0QQ, UK.
authors.forename: Alessandra
authors.initials: A
authors.lastname: De Riva
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Deery
authors.forename: Sarah
authors.initials: S
authors.lastname: McDonald
authors.forename: Torben
authors.initials: T
authors.lastname: Lund
authors.forename: Robert
authors.initials: R
authors.lastname: Busch
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000938:Antigen-Presenting Cells / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D017666:Deuterium Oxide / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D008285:Major Histocompatibility Complex; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000378:metabolism*; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis / Q000378:metabolism
chemical_list: D010455:Peptides; D011506:Proteins; D017666:Deuterium Oxide
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