Reliability of Protein Abundance and Synthesis Measurements in Human Skeletal Muscle

Abstract

The repeatability of dynamic proteome profiling (DPP), which is a novel technique for measuring the relative abundance (ABD) and fractional synthesis rate (FSR) of proteins in humans, is investigated. LC–MS analysis is performed on muscle samples taken from male participants (n = 4) that consumed 4 × 50 mL doses of deuterium oxide (2H2O) per day for 14 days. ABD is measured by label‐free quantitation and FSR is calculated from time‐dependent changes in peptide mass isotopomer abundances. One‐hundred one proteins have at least one unique peptide and are used in the assessment of protein ABD. Fifty‐four of these proteins meet more stringent criteria and are used in the assessment of FSR data. The median (M), lower‐, (Q1) and upper‐quartile (Q3) values for protein FSR (%/d) are M = 1.63, Q1 = 1.07, and Q3 = 3.24, respectively. The technical CV of ABD data has a median value of 3.6% (Q1 1.7% to Q3 6.7%), whereas the median CV of FSR data is 10.1% (Q1 3.5% to Q3 16.5%). These values compare favorably against other assessments of technical repeatability of proteomics data, which often set a CV of 20% as the upper bound of acceptability.

Fractional Synthesis Rates of Individual Proteins in Rat Soleus and Plantaris Muscles

“…Conversely, turnover of haemoglobin subunit beta 2 (HBB2) was 1.25%/day ± 0.14%/day in the current work and 4.74%/day ± 0.7%/day in [20]. FSR data exhibits greater biological variability than protein abundance data [19] but these inter-study differences may also relate to differences in rat strain or the analytical method used to calculate FSR. Holwerda et al [20], employed a non-linear calculation consistent with the two-point model (Figure 3) that uses data collected at the start and end of the labelling period only.…”

“…We report that individual proteins exhibit a broad range of turnover rates within skeletal muscle, but our analysis primarily focuses on proteins that are common to both soleus and plantaris muscle. To date, at least 17 papers (Table 2) have reported protein-specific FSR data in various muscles of humans [16,19,27,28], rodents [13,18,20,23,24,[29][30][31][32][33][34][35] and chickens [17] in vivo. The earliest works (e.g., [16,23,30]) used biochemical techniques to isolate abundant individual proteins.…”

“…Soluble proteins were processed for mass spectrometry analysis by in-solution digestion according to previous work from our laboratory [ 19 ]. Briefly, lysates containing 200 μg of protein were precipitated in 5 volumes of acetone at −20 °C overnight and then resuspended in UA buffer (8 M urea in 0.1 M Tris-HCl, pH 8.5).…”

“…The use of a time series experiment allows non-linear changes in D 2 O incorporation to be observed. Herein, data were analysed by semi-log plot and peptides with poor fitting (R 2 < 0.85) data were excluded from further analysis, consistent with our recent work [19].…”

“…Infusion of stable isotope-labelled amino acids constrains studies to a relatively short duration (e.g., min-h timescales), whereas the turnover of abundant tissue proteins in vivo occurs over a period of days. Longer-term labelling studies have been conducted in animals fed a diet enriched with a stable-isotope-labelled amino acid [13,17], whereas we have focused on the application of deuterium oxide (D 2 O or 2 H 2 O) or "heavy water" [14,[18][19][20][21]. D 2 O can be administered via the drinking water in free-living animals and the majority of amino acids are labelled intracellularly via transamination reactions [22].…”