Acetylated proteins in Human Embryonic Kidney cell line-293 (HEK-293) have been identified using nano-HPLC/tandem mass spectrometry for the first time at George Mason University. Hyperacetylation was induced by treating cells with the DNA alkylating agent methyl methanesulfonate (MMS) and an inhibitor of histone deacetylases, Trichostatin A (TSA). Cellular proteins were trypsin digested and immunoprecipitated with an anti-acetylated lysine antibody and analyzed by mass spectrometry. Acetylated proteins were compared across MMS+TSA treated, MMS treated, and control samples to identify acetylations which occurred in response to DNA alkylation. Seven candidate proteins were chosen based on spectral counts and their biological connection to DNA damage and their presence was verified either by in-gel digestion/MS/MS or western blot. The results of this study suggest that protein acetylation is an important cellular response to DNA damage.