Accurate quantification of site-specific acetylation stoichiometry reveals the impact of sirtuin deacetylase CobB on the E. coli acetylome

Lysine acetylationis a protein posttranslational modification(PTM) that occurs on thousands of lysine residues in diverse organisms from bacteria to humans. Accurate measurement of acetylation stoichiometryon a proteome-wide scale remains challenging. Most methods employ a comparison of chemically acetylatedpeptides to native acetylatedpeptides, however, the potentially large differences in abundance between these peptides presents a challenge for accurate quantification. Stable isotope labelingby amino acidsin cell culture(SILAC)-based mass spectrometry (MS) is one of the most widely used quantitative proteomicmethods. Here we show that serial dilutionof SILAC-labeled peptides (SD-SILAC) can be used to identify accurately quantified peptides and to estimate the quantification error rate. We applied SD-SILAC to determine absolute acetylation stoichiometryin exponentially-growing and stationary-phase wild-type and Sirtuindeacetylase CobB-deficient cells. To further analyze CobB-regulated sites under conditions of globally increased or decreased acetylation, we measured stoichiometryin phophotransacetylase (ptaΔ) and acetate kinase(ackAΔ) mutant strains in the presence and absence of the Sirtuininhibitor nicotinamide. We measured acetylation stoichiometryat 3,669 unique sites and found that the vast majority of acetylationoccurred at a low stoichiometry. Manipulations that cause increased nonenzymatic acetylationby acetyl-phosphate (AcP), such as stationary-phase arrest and deletion of ackA, resulted in globally increased acetylation stoichiometry. Comparison to relative quantification under the same conditions validated our stoichiometryestimates at hundreds of sites, demonstrating the accuracy of our method. Similar to Sirtuindeacetylase 3 ( SIRT3) in mitochondria, CobBsuppressed acetylationto lower than median stoichiometryin WT, ptaΔ, and ackAΔ cells. Together, our results provide a detailed view of acetylation stoichiometryin E. coli and suggest an evolutionarily conserved function of Sirtuindeacetylases in suppressing low stoichiometry acetylation.