Dynamic Profiling of Protein Mole Synthesis Rates During C2C12 Myoblast Differentiation

Mole (MSR) and fractional (FSR) synthesis rates of proteins during C2C12 myoblast differentiation were investigated. Myoblast cultures supplemented with D2O during 0–24 h or 72–96 h of differentiation were analysed by LC‐MS/MS to calculate protein FSR and MSR after samples were spiked with yeast alcohol dehydrogenase (ADH1). Profiling of 153 proteins detected 70 significant (p ≤ 0.05, FDR ≤1 %) differences in abundance between cell states. Early differentiation was enriched by clusters of ribosomal and heat shock proteins, whereas later differentiation was associated with actin filament binding. The median (first ‐ third quartile) FSR (%/h) during early differentiation 4.1 (2.7‐5.3) was ∼2‐fold greater than later differentiation 1.7 (1.0‐2.2), equating to MSR of 0.64, (0.38‐1.2) and 0.28, (0.1‐0.5) fmol/h/ug total protein, respectively. MSR corresponded more closely with abundance data and highlighted proteins associated with glycolytic processes and intermediate filament protein binding that were not evident amongst FSR data. Similarly, MSR during early differentiation accounted for 78 % of the variation in protein abundance during later differentiation, whereas FSR accounted for 4%. Conclusively, the interpretation of protein synthesis data differed when reported in mole or fractional terms, which has consequences when studying the allocation of cellular resources