Lysine acetylation is a common protein posttranslational modification that regulates a variety of biological processes. A major bottleneck to fully understanding the functional aspects of lysine acetylation is the difficulty in measuring the proportion of lysine residues that are acetylated. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of protein lysine acetylation. Using this technique, we determined the modification occupancy for ~750 acetylated peptides from mammalian cell lysates. Furthermore, the acetylation on N-terminal tail of histone H4 was cross-validated by treating cells with sodium butyrate, a potent deacetylase inhibitor, and comparing changes in stoichiometry levels measured by our method with immunoblotting measurements. Of note we observe that acetylation stoichiometry is high in nuclear proteins, but very low in mitochondrial and cytosolic proteins. In summary, our method opens new opportunities to study in detail the relationship of lysine acetylation levels of proteins with their biological functions. 1. Introduction Lysine acetylation (KAc) of proteins is a ubiquitous posttranslational modification (PTM) that controls many cellular processes. The dynamic regulation of KAc by lysine acetyltransferases (KATs) and deacetylases (KDACs) modulates many important cellular functions, such as cell metabolism and gene expression [1, 2]. Recent advances in mass spectrometry combined with immunoaffinity purification are enabling the identification and relative quantification of thousands of acetylation sites in a single experiment [3–5]. These new data have boosted the discovery of regulatory functions of KAc for many proteins, including a variety of metabolic enzymes . Although significant progress has been made, a major remaining hurdle in the field is the determination of acetylation stoichiometry on proteins. The knowledge of KAc stoichiometry is considered essential to better understand the mechanism and impact of this modification on the control protein functions, such as enzymatic activity [2, 6]. Indeed, this problem is not exclusive to KAc as there are almost no systematic determinations of the stoichiometry of PTMs. This has remained a challenge because methods to determine stoichiometry of PTMs are not compatible with enrichment procedures, since both modified and unmodified versions of polypeptides need to be present in the sample. Global studies have been successfully performed to determine the stoichiometries
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