LAUSR

Post‐translational modifications (PTMs) dynamically regulate proteins and biological pathways, typically through the combined effects of multiple PTMs. Lysine residues are targeted for various PTMs, including malonylation and succinylation. However, PTMs offer specific challenges to mass spectrometry‐based proteomics during data acquisition and processing. Thus, novel and innovative workflows using data‐independent acquisition (DIA) ensure confident PTM identification, precise site localization, and accurate and robust label‐free quantification. In this study, we present a powerful approach that combines antibody‐based enrichment with comprehensive DIA acquisitions and spectral library‐free data processing using directDIA (Spectronaut). Identical DIA data can be used to generate spectral libraries and comprehensively identify and quantify PTMs, reducing the amount of enriched sample and acquisition time needed, while offering a fully automated workflow. We analyzed brains from wild‐type and Sirtuin 5 (SIRT5)‐knock‐out mice, and discovered and quantified 466 malonylated and 2211 succinylated peptides. SIRT5 regulation remodeled the acylomes by targeting 164 malonylated and 578 succinylated sites. Affected pathways included carbohydrate and lipid metabolisms, synaptic vesicle cycle, and neurodegenerative diseases. We found 48 common SIRT5‐regulated malonylation and succinylation sites, suggesting potential PTM crosstalk. This innovative and efficient workflow offers deeper insights into the mouse brain lysine malonylome and succinylome.