Dynamic Profiling of Protein Mole Synthesis Rates during C2C12 Myoblast Differentiation

Mole (MSR) and fractional (FSR) synthesis rates of proteins during C2C12 myoblast differentiation are investigated. Myoblast cultures supplemented with D₂ O during 0-24 h or 72-96 h of differentiation are analyzed by LC-MS/MS to calculate protein FSR and MSR after samples are 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 is enriched by clusters of ribosomal and heat shock proteins, whereas later differentiation is associated with actin filament binding. The median (first-third quartile) FSR (%/h) during early differentiation 4.1 (2.7-5.3) is approximately twofold 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^-1  µg^-1 total protein, respectively. MSR corresponds more closely with abundance data and highlights proteins associated with glycolytic processes and intermediate filament protein binding that are not evident among FSR data. Similarly, MSR during early differentiation accounts for 78% of the variation in protein abundance during later differentiation, whereas FSR accounts for 4%. Conclusively, the interpretation of protein synthesis data differs when reported in mole or fractional terms, which has consequences when studying the allocation of cellular resources.