We used acetyl-lysine antibody-based affinity enrichment followed by mass spectrometry to identify acetylation changes caused by SIRT6 overexpression. We co-overexpressed Flag-OVOL2 and pCMV-SIRT6 in N2a cells. At 96 h after transfection, we treated cells with TSA for 12 h, collected proteins and digested proteins into peptides, enriched acetylated peptides using an anti-acetyl-lysine antibody, and then performed LC-MS/MS.