A New Method to Directly Quantify Acetylated Lysine Residues♦

Stoichiometry of Site-specific Lysine Acetylation in an Entire Proteome
    Open AccessPublished:August 01, 2014DOI:
        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.”