title: Bioorthogonal chemical reporters for monitoring protein acetylation.
abstract: Protein acetylation is a key post-translational modification that regulates diverse biological activities in eukaryotes. Here we report bioorthogonal chemical reporters that enable direct in-gel fluorescent visualization and proteome-wide identification of acetylated proteins via Cu(I)-catalyzed azide-alkyne cycloaddition, often termed "click chemistry". We demonstrate that two alkynyl-acetyl-CoA analogues, 4-pentynoyl-CoA and 5-hexynoyl-CoA, function as efficient substrates of lysine acetyltransferase p300 and serve as sensitive reagents for monitoring p300-catalyzed protein acetylation in vitro. In addition, we demonstrate that three alkynylacetate analogues, sodium 3-butynoate, sodium 4-pentynoate, and sodium 5-hexynoate, can be metabolically incorporated onto cellular proteins through biosynthetic mechanisms for profiling of acetylated proteins in diverse cell types. Mass spectrometry analysis of the enriched 4-pentynoate-labeled proteins revealed many reported as well as new candidate acetylated proteins from Jurkat T cells and specific sites of lysine acetylation. The bioorthogonal chemical reporters described here should serve as powerful tools for investigating protein acetylation.
authors.affiliation: Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York 10065, USA.
authors.forename: Yu-Ying
authors.initials: YY
authors.lastname: Yang
authors.forename: Janice M
authors.initials: JM
authors.lastname: Ascano
authors.forename: Howard C
authors.initials: HC
authors.lastname: Hang
mesh_terms: D000107:Acetylation; D000480:Alkynes / Q000737:chemistry; D000818:Animals; D001386:Azides / Q000737:chemistry; D002384:Catalysis; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation*; D003300:Copper / Q000737:chemistry; D006801:Humans; D019169:Jurkat Cells; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D000480:Alkynes; D001386:Azides; D011506:Proteins; D003300:Copper