Analysis of human acetylation stoichiometry defines mechanistic constraints on protein regulation.

Lysine acetylation is a reversible posttranslational modification that occurs at thousands of sites on human proteins. However, the stoichiometry of acetylation remains poorly characterized, and is important for understanding acetylation-dependent mechanisms of protein regulation. Here we provide accurate, validated measurements of acetylation stoichiometry at 6829 sites on 2535 proteins in human cervical cancer (HeLa) cells. Most acetylation occurs at very low stoichiometry (median 0.02%), whereas high stoichiometry acetylation (>1%) occurs on nuclear proteins involved in gene transcription and on acetyltransferases. Analysis of acetylation copy numbers show that histones harbor the majority of acetylated lysine residues in human cells. Class I deacetylases target a greater proportion of high stoichiometry acetylation compared to SIRT1 and HDAC6. The acetyltransferases CBP and p300 catalyze a majority (65%) of high stoichiometry acetylation. This resource dataset provides valuable information for evaluating the impact of individual acetylation sites on protein function and for building accurate mechanistic models.

Accurate Quantification of Site-specific Acetylation Stoichiometry Reveals the Impact of Sirtuin Deacetylase CobB on the E. coli Acetylome.

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.