Lysine acetylation is a protein posttranslational modification (PTM) that occurs 
on thousands of lysine residues in diverse organisms from bacteria to humans. 
Accurate measurement of acetylation stoichiometry on a proteome-wide scale 
remains challenging. Most methods employ a comparison of chemically acetylated 
peptides to native acetylated peptides, however, the potentially large 
differences in abundance between these peptides presents a challenge for 
accurate quantification. Stable isotope labeling by amino acids in cell culture 
(SILAC)-based mass spectrometry (MS) is one of the most widely used quantitative 
proteomic methods. Here we show that serial dilution of 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 stoichiometry in exponentially-growing and stationary-phase 
wild-type and Sirtuin deacetylase CobB-deficient cells. To further analyze 
CobB-regulated sites under conditions of globally increased or decreased 
acetylation, we measured stoichiometry in phophotransacetylase (ptaΔ) and 
acetate kinase (ackAΔ) mutant strains in the presence and absence of the Sirtuin 
inhibitor nicotinamide. We measured acetylation stoichiometry at 3,669 unique 
sites and found that the vast majority of acetylation occurred at a low 
stoichiometry. Manipulations that cause increased nonenzymatic acetylation by 
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 stoichiometry estimates 
at hundreds of sites, demonstrating the accuracy of our method. Similar to 
Sirtuin deacetylase 3 (SIRT3) in mitochondria, CobB suppressed acetylation to 
lower than median stoichiometry in WT, ptaΔ, and ackAΔ cells. Together, our 
results provide a detailed view of acetylation stoichiometry in E. coli and 
suggest an evolutionarily conserved function of Sirtuin deacetylases in 
suppressing low stoichiometry acetylation.