A Temporal Phospho-acetylome Atlas of Human Myogenesis Identifies coordinated Post-Translational Regulation

Skeletal muscle differentiation depends on precise temporal regulation of protein modifications. To define how phosphorylation and lysine acetylation change during this process, we applied tandem mass tag (TMT)-based quantitative proteomics to human myoblasts sampled at six stages spanning proliferation, induction of differentiation, and early myotube formation. Using sequential enrichment of phosphorylated and acetylated peptides, high-pH fractionation, and high-resolution mass spectrometry, we identified more than 22,000 modified peptides and quantified their temporal behavior after correction for protein abundance. Phosphorylation exhibited extensive site-specific remodeling throughout the time course, whereas acetylation showed a pronounced relative increase during late differentiation. Integration of corrected modification levels with protein abundances and temporal clustering revealed stage-specific regulation of processes linked to cell-cycle withdrawal, metabolic transitions, cytoskeletal reorganization, and chromatin-associated functions. Predicted temporal activity profiles of kinases, acetyltransferases, and deacetylases uncovered coordinated regulatory patterns, including activity relationships involving CSNK2A1-HDAC1/2, PRKAA1-HAT1, and CDK1/2-KAT7. Dual-modified proteins such as lamin A/C and glycolytic enzymes display densely regulated clusters of phosphorylation and acetylation sites that may contribute to nuclear remodeling and metabolic adaptation during myogenesis. Together, this work provides a high-resolution temporal phospho-acetylome atlas of human muscle cell differentiation, identifies candidate phosphorylation-acetylation coordination patterns, and establishes a systems-level resource for future mechanistic studies of post-translational regulation in skeletal muscle development.