Turnover rates of human muscle proteins in vivo reported in fractional, mole and absolute units

Protein fractional turnover rates (FTR) represent measurements of flux through a protein pool, i.e., net abundance (ABD) of the protein. However, if protein abundance is not measured or varies between experimental conditions, the interpretation of FTR data may be confounded, potentially leading to misleading conclusions about protein dynamics. This project investigates the consequences of reporting turnover rates of human muscle proteins in vivo using mole and absolute units (which incorporate protein abundance data) compared to fractional (%/d) data, which do not account for abundance. By providing a comprehensive comparison of these metrics, we aim to refine the interpretation of protein turnover studies and offer a methodological framework for future research. Three physically active males (21 ± 1 years) were recruited and underwent a 12-day protocol of daily deuterium oxide (D2O) consumption, with vastus lateralis biopsies collected on days 8 and 12. Protein abundances were normalized to yeast alcohol dehydrogenase, added during sample preparation, to ensure consistency across samples. Fractional turnover rates were calculated based on time-dependent changes in peptide mass isotopomer profiles. To enable a more physiologically relevant interpretation of turnover, FTR data were combined with abundance measurements (fmol/μg protein) to calculate mole turnover rates (MTR; fmol/μg protein/d) and absolute turnover rates (ATR; ng/μg protein/d), providing a more complete representation of protein kinetics. Abundance data were collected for 1,772 proteins and FTR data were calculated from 3,944 peptides representing 935 proteins (average 3 peptides per protein). The median (M), lower- (Q1) and upper-quartile (Q3) values for protein FTR (%/d) were M = 4.3, Q1 = 2.52, Q3 = 7.84. Our analyses suggest that MTR provides a more informative metric than FTR, particularly for studies investigating multiprotein complexes, where MTR accounts for potential differences in the molecular weights of component subunits. ATR may be even more advantageous than MTR and FTR, as it facilitates comparisons between samples with different abundance profiles, making it a preferred metric in conditions where protein abundance varies across experimental groups. These findings highlight the importance of incorporating protein abundance into turnover calculations to improve the biological relevance and interpretability of turnover data.