Lysine acetylation is a major posttranslational modification. Acetylation is implicated in diverse cellular functions, most prominently including the epigenetic regulation of gene transcription. Our laboratory is using mass spectrometry (MS)-based quantitative proteomics to map the scope of acetylation as well as to investigate its dynamic regulation in response to genetic and environmental perturbations. Furthermore, we developed novel proteomic methods to accurately quantify the site-specific stoichiometry of acetylation on a proteome-wide scale. Our results show that acetylation can occur through both enzymatic and non-enzymatic mechanisms, and enzyme-catalyzed acetylation appears to have a higher stoichiometry. I will discuss our recently published and ongoing efforts in understanding acetylation signaling.