Stoichiometry of Site-specific Lysine Acetylation in an Entire Proteome

• Published in: The Journal of biological chemistry Vol. 289; no. 31; pp. 21326 - 21338
• Main Authors: Baeza, Josue, Dowell, James A., Smallegan, Michael J., Fan, Jing, Amador-Noguez, Daniel, Khan, Zia, Denu, John M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2014; American Society for Biochemistry and Molecular Biology
• Subjects: Acetyl-CoA Synthetase; Acetylation; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Chromatography, High Pressure Liquid; Computational Biology; Escherichia coli (E. coli); Escherichia coli - metabolism; Genomics and Proteomics; Lysine - metabolism; Mass Spectrometry (MS); Metabolism; Molecular Sequence Data; Proteome; Proteomics; Stoichiometry; Tandem Mass Spectrometry; Stoichiometry; Escherichia coli (E. coli); Acetyl-CoA Synthetase; Proteomics; Mass Spectrometry (MS); Acetylation; Metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.581843

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. Background: Lysine acetylation sites have been mapped, but information on stoichiometry is lagging. Results: We developed and utilized the first direct, unbiased method for quantifying site-specific acetylation stoichiometry of a proteome without antibody enrichment. Conclusion: High stoichiometry is associated with central metabolism, transcription, and translation. Loss of deacetylase CobB affects site-specific and global acetylation stoichiometry, altering acetyl-CoA metabolism. Significance: Stoichiometry provides functional insight into protein acetylation.