title: Site-Specific Identification of Lysine Acetylation Stoichiometries in Mammalian Cells.
abstract: Functional characterization of the lysine acetylation pathway requires quantitative measurement of the modification abundance at the stoichiometry level. Here, we developed a systematic workflow for global untargeted identification of site-specific Lys acetylation stoichiometries in mammalian cells. Our strategy includes an optimized protocol for in vitro chemical labeling of unmodified lysine with stable isotope-encoded acetyl-NHS ester, deep proteomic profiling with a high resolution mass spectrometer, and a new software tool for quantitative analysis and stoichiometry determination. The workflow was validated using in vitro chemically labeled BSA and synthetic peptides with multiple Lys acetylations at various positions. In the proof-of-concept study, we applied the strategy to analyze the proteome of HeLa cells and determined the stoichiometries of over 600 acetylation sites with good reproducibility. Sodium butyrate treatment induced a significant increase of acetylation stoichiometries in HeLa cells. Analysis of site-specific stoichiometry dynamics revealed the coregulation of closely positioned acetylation sites on histones H3 and H4 upon treatment.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota at Twin Cities , Minneapolis, Minnesota 55455, United States.
authors.forename: Tong
authors.initials: T
authors.lastname: Zhou
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota at Twin Cities , Minneapolis, Minnesota 55455, United States.
authors.forename: Ying-Hua
authors.initials: YH
authors.lastname: Chung
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota at Twin Cities , Minneapolis, Minnesota 55455, United States.
authors.forename: Jianji
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota at Twin Cities , Minneapolis, Minnesota 55455, United States.
authors.forename: Yue
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000107:Acetylation; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics; D015203:Reproducibility of Results
chemical_list: D006657:Histones; D020543:Proteome; D008239:Lysine
keywords: lysine acetylation; quantitative proteomics; stoichiometry