Lysine acetylation levels were compared in wt and an acetate kinase (ackA) mutant in Neisseria gonorrhoeae. Acetylated peptides were enriched for using affinity enrichment and were compared using label-free MS-1 quantification. Over a hundred acetylated lysine sites were shown to be regulated. A number of these sites were found to be involved in bacterial metabolism and some were found in proteins known to or predicted to play a role in pathogenesis.