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 (2H2O), a convenient, universal biosynthetic label, for measuring protein synthesis. Analyses of 2H-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 2H2O labeling, as applied to various proteins of the major histocompatibility complex (MHC). Human and murine antigen-presenting cells were cultured in medium containing 5% 2H2O; 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 2H2O 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.

Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling

“…To determine the extent to which the precursor proteogenic amino acid pool is labeled, after an intraperitoneal injection of 2 H 2 O, 2 H labeling of hepatic free amino acids at various time points was assessed by GC-MS (Table I) (12,15,17), alanine, glutamate, glutamine, glycine, and serine were extensively labeled, reflecting their central role in intermediary metabolism. Small, in some cases negligible, amounts of 2 H label were found in most essential amino acids.…”

“…Recently the global metabolic labeling of the entire proteome was achieved using 15 N-labeled medium and diet for cell culture and animal studies, respectively (9,10). Although these techniques allow estimation of global proteome dynamics without the requirement of precursor isotope abundance, these approaches require a long term time course experiment for exponential fitting.…”

“…Although these techniques allow estimation of global proteome dynamics without the requirement of precursor isotope abundance, these approaches require a long term time course experiment for exponential fitting. In addition, these techniques are not suited for human studies because they require the consumption of a fully 15 N-labeled diet. Recently we and others developed the 2 H 2 O metabolic labeling technique to measure protein turnover in free living animals (11)(12)(13)(14)(15).…”

“…In addition, these techniques are not suited for human studies because they require the consumption of a fully 15 N-labeled diet. Recently we and others developed the 2 H 2 O metabolic labeling technique to measure protein turnover in free living animals (11)(12)(13)(14)(15). In rats that have been given intraperitoneal injection of 2 H 2 O, 2 H equilibrates with total body water and amino acids such as alanine within 10 and 20 min, respectively (16) (Fig.…”

Metabolic Labeling Reveals Proteome Dynamics of Mouse Mitochondria

“…Isotope tracers are particularly useful for tracking such continual renewal of the proteome in living systems, because they allow differentiation between preexisting and newly synthesized proteins (5 (11)(12)(13)(14) and peptide analysis in MALDI-TOF MS (15) and LC-MS (16,17). More recently, Price et al described an approach for measuring protein turnover by calculating the theoretical number of 2 H-labeling sites on a peptide sequence (18) and reported the turnover rates of ϳ100 human plasma proteins.…”