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.

Fig. 1

Application of SINEW with peptide LC–MS to cultured antigen presenting cell lines. (A) Biosynthetic labeling of nonessential amino acids of proteins from ²H₂O. (B) Flow chart of experimental approach. (C) ²H₂O labeling does not perturb cell growth. Growth kinetics of Priess cells are shown during 1 week of culture in medium with or without 4.5% ²H₂O. Exponential curve fits and doubling times (t₂) are shown. (D) Constant ²H₂O enrichment in medium throughout the culture period (MPE, mole per cent excess over natural abundance) was verified by IRMS analysis of medium sampled at different times. (E–G) Protein isolation. HLA-DR (E), HLA-A/B/C (F), and H2-A (G) molecules were immunoprecipitated from host cell lines (Priess in panels E and F, M12.NOD in panel G) using appropriate mAbs: L243, W6/32, and OX-6, respectively (lanes 2). Lanes 1 represent control immunoprecipitates using irrelevant control antibody (Mac4 in panels E and F, MKD6 in panel G). Lanes 3 represent control immunoprecipitates from irrelevant cell lines (A20 cells in panels E and F). Samples were analyzed by nonreducing 12% SDS–PAGE and visualized by Coomassie blue staining. Positions of bands representing immunoprecipitating antibody, MHC class II α- and β-chains, MHC class I heavy chain (HC), and β₂-microglobulin (β₂m) are indicated. Under nonreducing conditions, the bands migrated slightly faster than the nominal molecular weights for each polypeptide (35 and 29 kDa for class II α- and β-chains, respectively, and 45 kDa for class I heavy chains; MW markers not shown).

Fig. 2

Identification of peptides suitable for ²H₂O labeling studies. (A and B) Identification of Ala-/Gly-containing HLA-DR4-derived tryptic peptides. DRα (A) and DRβ (B) bands excised from SDS gels of L243 immunoprecipitates from Priess cell extracts were reduced, carbamidomethylated, digested with trypsin, and analyzed by LC–MS/MS. Alignments of tryptic fragments to the sequence of their parent polypeptides are shown. Peptides selected for analysis are shown in bold type and underlined. (C and D) LC–MS analysis of mass isotopomer distributions for the DRα peptide, FASFEAQGALANIAVDK. (C) LC chromatogram of a DRα digest, with SIM for m/z = 876.45, corresponding to the M0 mass isotopomer of the doubly charged peptide. Integration of the principal LC peak for this mass isotopomer, and for higher order mass isotopomers, was used to quantify mass isotopomer distributions. (D) Mass spectrum for the LC peak of the intact DRα peptide showing a single, well-resolved set of mass isotopomers.

Fig. 3

Mass isotopomer distributions of HLA-DR4-derived tryptic peptides. (A–D) Analysis of DRα (51–67) peptide. (A) MIDA model and experimental quantification (means ± SDs of N = 6 replicate injections) of mass isotopomer distributions of the unlabeled DRα peptide. (B) Mass spectrum of the DRα peptide obtained before and after 6 days of culturing Priess cells in 4.5% ²H₂O. (C) MIDA model and experimental quantification (means ± SDs, N = 5) of mass isotopomer distributions after ²H₂O labeling. The model assumes that n = 22 labeling sites are accessible to label without dilution of the precursor pool enrichment (p) relative to the ²H₂O enrichment in medium; the value of n was adjusted for optimal fit to the data. (D) The difference between the mass isotopomer distributions of the fully labeled and unlabeled DRα peptide is shown. The inset shows the RMSD of the experimental data from a series of models in which p was kept at 4.5% and n was varied. The lowest RMSD value was obtained at n = 22. (E–H) The same analysis was applied to the DR0401β (73–80) peptide: (E) unlabeled mass isotopomer distributions (means ± SDs, N = 5 replicates); (F) mass spectra of unlabeled versus extensively labeled peptide; (G) mass isotopomer distributions of labeled peptide (means ± SDs of N = 5 replicate analyses, RMSD = 0.45% vs. model with n = 8 labeling sites and p = 4.5%); (H) difference analysis and dependence of model fit on n (inset).

Fig. 4

Measurement of fractional MHC protein synthesis. (A–D) Analysis of H2-Aα peptide obtained from A^g7 immunoprecipitates after varying times of continuous labeling of M12.NOD cells. (A) Mass isotopomer distributions after different times of labeling. Note the intersection of all mass spectra at an iso-abundant point near M2 (arrow). (B) Evolution of fractional mass isotopomer abundances from their baseline values as a function of labeling time. Each mass isotopomer shifted from its baseline value to its fully labeled abundance with the same kinetics, reflecting the rate of fractional protein synthesis. (C) Convergent estimates of fractional H2-A^g7 protein synthesis as a function of labeling time obtained from analysis of different mass isotopomers of the Aα peptide. (D) Indistinguishable estimates of fractional H2-A^g7 protein synthesis obtained from analysis of α- and β-chain peptides. Data were averaged for the different mass isotopomers within each peptide (excluding those that exhibited experimental noise due to small abundance shifts such as M2 in the α-chain peptide). Error bars represent SDs and in most instances are smaller than the symbols. Similar analyses were performed for α- and β-chain peptides of H2-A^d molecules isolated from A20 cells (E) and for HLA-DR4 molecules isolated from Priess cells (F). (G and H) The indicated peptides specific for the HLA-B8 (G) and HLA-B51 (H) alleles were analyzed after W6/32 immunoprecipitation from LCL721 cells. In panel G, two charge states of the same peptide were analyzed. In panel H, two peptides were analyzed, and one of these was present in two charge states, as indicated.