title: Site-Specific Quantification of Lysine Acetylation Using Isotopic Labeling.
abstract: Acetylation of ɛ-amino group of lysine is one of the most common protein posttranslational modifications. The modification is reversible and catalyzed by lysine acetyltransferases in one direction and lysine deacetylases in the other direction. Although numerous lysine acetylation sites have been identified in many proteins involved in a diverse range of cellular processes, little has been revealed about the roles of this modification at the level of individual sites. To understand better the site-specific roles of this modification, it is important to investigate what fraction of each modified site is actually acetylated (stoichiometry of acetylation) in vivo in different physiological conditions. Here we describe a method that allows us to determine the site-specific stoichiometry of lysine acetylation. The method chemically acetylates all of the lysine residues in proteins that are not endogenously acetylated with an isotopically labeled acetyl (13C2-acetyl) group. The chemical treatment enables to determine the stoichiometry of acetylation at individual sites by measuring the abundance of the endogenously acetylated group (carrying a natulally abundant12C2-acetyl group) and the chemically introduced acetyl group (carrying an isotopically labeled13C2-acetyl group) in the subsequent mass spectrometry analysis. The method is most suitable to apply to pure proteins or relatively simple protein mixtures.
authors.affiliation: Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, OH, United States. Electronic address: masaru.miyagi@case.edu.
authors.forename: M
authors.initials: M
authors.lastname: Miyagi
mesh_terms: D000089:Acetic Anhydrides / Q000737:chemistry; D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002247:Carbon Isotopes / Q000737:chemistry; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006657:Histones / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D007553:Isotope Labeling; D008239:Lysine / Q000737:chemistry*; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000302:isolation & purification; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D000089:Acetic Anhydrides; D002247:Carbon Isotopes; D006657:Histones; D024101:Mitochondrial Proteins; C031800:acetic anhydride; D008239:Lysine
keywords: Acetylation; Isotope labeling; LC-MS/MS; Mass spectrometry; Proteomics; Stoichiometry