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

Abstract

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.