Mass spectrometric workflow.

(A) Three biological replicates of N. gonorrhoeae strains 1291wt and 1291ackA were grown overnight in broth cultures. Cells were harvested, washed, and a protein lysate was generated and digested with trypsin. Affinity enrichment for acetyl-lysine (K-acetyl)-containing peptides was performed using a polyclonal anti-acetyl-Lys antibody. Enriched K-acetyl peptides were analyzed by high-resolution label-free LC-MS/MS and quantified using MS1 quantitation. (B) An example of a peptide analyzed by MS1 quantitation using Skyline. MS1 ion chromatograms of the precursor ions for M, M+1, and M+2 for the acetylated peptide IKLDK*LVSEGFER, at K-329, from the zinc-binding alcohol dehydrogenase protein for 1291wt and 1291ackA are shown. MS1 quantitation was performed by extracting and measuring the peak areas from the most intense isotopic precursors and subsequently comparing the wt and ackA mutant peptides.