Dynamic Proteome Profiling of Protein Fractional and Molar Synthesis Rates in Human Muscle in Vivo

This study aimed to develop a method for measuring the abundance and synthesis rate of human muscle proteins in molar rather than fractional (%/d) terms. Losses in proteostasis are implicated in ageing and chronic diseases but research is hindered by a lack of methods for studying protein dynamics in humans. Protein‐specific fractional synthesis rate (FSR) can be measured by stable isotope labelling and peptide mass spectrometry (MS). However, FSR is a measurement of an unknown whole, i.e. protein abundance. If protein abundance changes or is different between conditions, the interpretation of FSR data may be confounded.Three physically active males (21 ± 1 years; height 178 ± 1 cm; weight 75 ± 5 kg) gave their informed consent to the ethically approved procedures. Newly synthesised proteins were labelled by oral consumption of deuterium oxide (50 ml of 99.8 atom %) taken four times per day during days 1–5, and two times per day during days 6–12. Samples of blood and vastus lateralis were collected after an overnight fast on days 0, 4, 8 and 12. Precursor enrichment and incorporation of deuterium oxide into muscle proteins were determined by liquid chromatography tandem MS. Protein abundances were normalised to 30 fmol yeast alcohol dehydrogenase added during sample preparation. FSR (%/d) was calculated from time‐dependent changes in peptide mass isotopomer abundances. FSR and abundance data (fmol/ μg protein) were combined to calculate molar synthesis rates (MSR; fmol/ μg protein/ d).Abundance data were collected for 1,368 proteins that had at least 1 unique peptide. FSR data were calculated from 3,944 peptides representing 940 proteins (average 3 peptides per protein, range 1 – 53 peptides per protein). In total, 769 proteins had abundance and FSR data in two or more participants. The median (M), lower‐(Q1) and upper‐quartile (Q3) values for protein FSR (%/d) were M = 4.39, Q1 = 0.96, Q3 = 7.84. Alpha skeletal muscle actin had the lowest FSR (0.28 ± 0.1 %/d) whereas the highest recorded FSR was 54.43 ± 8.9 %/d (nuclear protein localization protein 4 homolog). tRNA ligases were common amongst top‐ranked proteins by FSR but were ranked amongst the lowest in abundance e.g. Leucine‐‐tRNA ligase was the lowest abundance (5 amol/ μg protein) protein detected. The most abundant protein was myoglobin (1.52 pmol/ μg protein). Average MSR (fmol/ μg protein/ d) was M = 0.05 (Q1 = 0.017, Q3 = 0.13). Metabolic enzymes were amongst the top‐ranked proteins by MSR (range 1–10 fmol/ μg protein/ d) alongside cullin‐associated NEDD8‐dissociated protein 2 (0.9 ± 0.2 fmol/ μg protein/ d) and antioxidant enzymes, including mitochondrial superoxide dismutase [Mn], peroxiredoxin 3, peroxiredoxin 2 and glutathione S‐transferase‐P (each ~0.5 fmol/ μg protein/ d).MSR proteomic data offers new insight to the allocation of cellular resources. Future applications investigating muscle adaptation or differences between health and disease may uncover unexpected insight regarding which proteins are most synthesised and highlight new avenues of research in human muscle physiology.