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A New Method to Directly Quantify Acetylated Lysine Residues♦

Stoichiometry of Site-specific Lysine Acetylation in an Entire Proteome
    Open AccessPublished:August 01, 2014DOI:https://doi.org/10.1074/jbc.P114.581843
        Figure thumbnail gr1
        Diagram of method for determining direct acetylation stoichiometry. Extracted protein was denatured, chemically acetylated using isotopic acetic anhydride followed by trypsin digestion. Peptides were analyzed by LC-MS/MS and quantified to determine site-specific stoichiometry.
        ♦ See referenced article, J. Biol. Chem. 2014, 289, 21326–21338
        Acetylation of the side-chain amino group on lysines is a protein post-translational modification that influences a number of cellular processes, such as protein-protein interactions, protein-DNA interactions, and enzymatic activity, in a variety of organisms. However, researchers don't have an easy way of measuring the levels of acetylation at specific lysine residues in a collection of proteins. In this Paper of the Week, John Denu at the University of Wisconsin-Madison and colleagues describe a new method for directly quantifying the stoichiometry of acetylated lysines in the proteome of Escherichia coli. The method relies on combining isotopic labeling of unacetylated lysine residues with a bioinformatic algorithm, bypassing the need for antibody enrichment, which complicates the analysis. Using their new approach, Denu and colleagues found that metabolic enzymes, which either use or make acetyl-CoA, and proteins involved in transcriptional and translational processes had a high fraction of acetylated lysine residues. The investigators also demonstrated that the loss of a NAD+-dependent protein deacetylase, CobB, increased global acetylation at low stoichiometry sites, caused site-specific changes at high stoichiometry sites, and altered acetyl-CoA metabolism. The authors concluded that a deficiency of CobB “leads to both site-specific and global changes in protein acetylation stoichiometry, affecting central metabolism.”