Abstract
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.
Highlights
Lysine acetylation is a reversible posttranslational modification that occurs at thousands of sites on human proteins
We required that the abundance of native acetylated peptides was quantified by comparison with at least two different concentrations of chemically acetylated peptides, and that the measured SILAC ratios agreed with the serial dilution series
As a general rule, the mechanisms by which acetylation regulates protein function should agree with a low stoichiometry of modification
Summary
Lysine acetylation is a reversible posttranslational modification that occurs at thousands of sites on human proteins. 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. Proteome-wide analyses of lysine acetylation should focus on identifying parameters that will help prioritize the functional relevance of individual sites and provide mechanistic insights These parameters include regulation by acetyltransferases and deacetylases, dynamic turnover rates, and the stoichiometry of modification. We validate our results using known quantities of peptide standards, using recombinant acetylated proteins, and by comparison with acetylated peptide intensity This high-confidence dataset is used to calculate acetylation copy numbers in cells, to explore the relationship between stoichiometry and regulation by acetyltransferases and deacetylases, and to reveal mechanistic constraints on protein regulation by acetylation
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