Cysteine S-acetylation is a widespread post-translational modification on metabolic proteins.

Protein acetylation is a fundamental regulatory mechanism occurring primarily on lysine amino acids. Here we report systematic in vivo characterization of cysteine S-acetylation as a widespread post-translational modification in mammalian tissues. By developing specialized sample preparation methods that preserve the labile thioester bond, we identified over 400 sites of cysteine acetylation in mouse liver, mirroring the abundance of lysine acetylation. Proteomic surveys across nine murine tissues revealed tissue-specific acetylation patterns that are enriched on metabolic enzymes in the cytoplasm. Cold exposure in mice triggers coordinated remodeling of the brown adipose tissue cysteine acetylome. Functional studies demonstrate that the acetylation of GAPDH Cys150 abolishes catalytic activity and correlates with nuclear enrichment, paralleling the known effects of S-nitrosylation on this enzyme. These findings establish cysteine acetylation as a widespread modification of metabolic proteins that responds to changes in cellular acetyl-CoA availability, fundamentally expanding the landscape of protein acetylation beyond lysine.