In this study, we conducted a comprehensive analysis to elucidate the overall pattern of three types of acidic lysine-acylation modifications within Staphylococcus aureus subsp. aureus ATCC 25923. By applying mass spectrometry techniques, we systematically investigated the effects of these lysine modifications on protein function. In our study, we identified 1,255 acetylation sites, 876 succinylation sites, and 67 malonylation sites, thereby contributing to the expansion of the lysine acylation modification network.