Numbers of Exchangeable Hydrogens from LC-MS Data of Heavy Water Metabolically Labeled Samples

Labeling with deuterium oxide (D₂O) has emerged as one of the preferred approaches for measuring the synthesis of individual proteins in vivo. In these experiments, the synthesis rates of proteins are determined by modeling mass shifts in peptides during the labeling period. This modeling depends on a theoretical maximum enrichment determined by the number of labeling sites (N_EH) of each amino acid in the peptide sequence. Currently, N_EH is determined from one set of published values. However, it has been demonstrated that N_EH can differ between species and potentially tissues. The goal of this work was to determine the number of N_EH for each amino acid within a given experiment to capture the conditions unique to that experiment. We used four methods to compute the N_EH values. To test these approaches, we used two publicly available data sets. In a de novo approach, we compute N_EH values and the label enrichment from the abundances of three mass isotopomers. The other three methods use the complete isotope profiles and body water enrichment in deuterium as an input parameter. They determine the N_EH values by (1) minimizing the residual sum of squares, (2) from the mole percent excess of labeling, and (3) the time course profile of the depletion of the relative isotope abundance of monoisotope. In the test samples, the method using residual sum of squares performed the best. The methods are implemented in a tool for determining the N_EH for each amino acid within a given experiment to use in the determination of protein synthesis rates using D₂O.