title: Stoichiometry of site-specific lysine acetylation in an entire proteome.
abstract: Acetylation of lysine ϵ-amino groups influences many cellular processes and has been mapped to thousands of sites across many organisms. Stoichiometric information of acetylation is essential to accurately interpret biological significance. Here, we developed and employed a novel method for directly quantifying stoichiometry of site-specific acetylation in the entire proteome of Escherichia coli. By coupling isotopic labeling and a novel pairing algorithm, our approach performs an in silico enrichment of acetyl peptides, circumventing the need for immunoenrichment. We investigated the function of the sole NAD(+)-dependent protein deacetylase, CobB, on both site-specific and global acetylation. We quantified 2206 peptides from 899 proteins and observed a wide distribution of acetyl stoichiometry, ranging from less than 1% up to 98%. Bioinformatic analysis revealed that metabolic enzymes, which either utilize or generate acetyl-CoA, and proteins involved in transcriptional and translational processes displayed the highest degree of acetylation. Loss of CobB led to increased global acetylation at low stoichiometry sites and induced site-specific changes at high stoichiometry sites, and biochemical analysis revealed altered acetyl-CoA metabolism. Thus, this study demonstrates that sirtuin deacetylase deficiency leads to both site-specific and global changes in protein acetylation stoichiometry, affecting central metabolism.
authors.affiliation: From the Department of Biomolecular Chemistry, the Wisconsin Institute for Discovery, and.
authors.forename: Josue
authors.initials: J
authors.lastname: Baeza
authors.affiliation: the Wisconsin Institute for Discovery, and.
authors.forename: James A
authors.initials: JA
authors.lastname: Dowell
authors.affiliation: the Wisconsin Institute for Discovery, and.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Smallegan
authors.affiliation: the Wisconsin Institute for Discovery, and.
authors.forename: Jing
authors.initials: J
authors.lastname: Fan
authors.affiliation: Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53715 and.
authors.forename: Daniel
authors.initials: D
authors.lastname: Amador-Noguez
authors.affiliation: the Center for Bioinformatics and Computational Biology, Department of Computer Science, University of Maryland, College Park, Maryland 20742.
authors.forename: Zia
authors.initials: Z
authors.lastname: Khan
authors.affiliation: From the Department of Biomolecular Chemistry, the Wisconsin Institute for Discovery, and jmdenu@wisc.edu.
authors.forename: John M
authors.initials: JM
authors.lastname: Denu
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D019295:Computational Biology; D004926:Escherichia coli / Q000378:metabolism; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D020543:Proteome; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D020543:Proteome; D008239:Lysine
keywords: Acetyl-CoA Synthetase; Acetylation; Escherichia coli (E. coli); Mass Spectrometry (MS); Metabolism; Proteomics; Stoichiometry