We developed an on-antibody tandem mass tag (TMT) labeling workflow for quantitative profiling of multiple lysine acylations and validated its reliability in Escherichia coli and Saccharomyces cerevisiae. The method enabled reproducible analysis of acetylation, propionylation, malonylation, and succinylation. Using this platform, we characterized dynamic acetylome changes in E. coli under temperature and pH perturbations, growth-cycle progression, and carbon-source alterations. We further identified AceA K308 and Ahr K127 as functional acetylation sites, with mutations reducing enzyme activity. This workflow provides a robust tool for studying lysine acylation and metabolic adaptation.