A protocol for detection of mitochondrial protein malonylation and succinylation

Recent advances in mass spectrometry and proteomics have revealed numerous novel lysine acylation modifications, which increase proteome functional diversity via covalent modification of lysine residues. Protein malonylation and succinylation are highly enriched in mitochondrial proteins. They use malonyl-CoA and succinyl-CoA which were primarily synthesized in mitochondria, as acyl donors and can modulate protein structure and function. However, conventional mass spectrometry-based workflows often suffer from limited coverage and may fail to detect low-abundance modification sites. Therefore, we present an organelle-centered proteomic protocol. By purifying mitochondria before enriching modified peptides, the protocol minimizes interference from non-mitochondrial proteins, thereby offering a valuable reference for investigating diverse protein acylation events in metabolic-associated fatty liver disease (MASLD) and other physiological and pathological contexts.