TY - JOUR T1 - Stoichiometry of site-specific lysine acetylation in an entire proteome. JF - J Biol Chem Y1 - 2014 A1 - Baeza, Josue A1 - Dowell, James A A1 - Smallegan, Michael J A1 - Fan, Jing A1 - Amador-Noguez, Daniel A1 - Khan, Zia A1 - Denu, John M KW - Acetylation KW - Amino Acid Sequence KW - Bacterial Proteins KW - Chromatography, High Pressure Liquid KW - Computational Biology KW - Escherichia coli KW - Lysine KW - Molecular Sequence Data KW - Proteome KW - Tandem Mass Spectrometry AB -

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.

VL - 289 CP - 31 M3 - 10.1074/jbc.M114.581843 ER -