title: Identification of lysine acetyltransferase p300 substrates using 4-pentynoyl-coenzyme A and bioorthogonal proteomics.
abstract: Proteomic studies have identified a plethora of lysine acetylated proteins in eukaryotes and bacteria. Determining the individual lysine acetyltransferases responsible for each protein acetylation mark is crucial for elucidating the underlying regulatory mechanisms, but has been challenging due to limited biochemical methods. Here, we describe the application of a bioorthogonal chemical proteomics method to profile and identify substrates of individual lysine acetyltransferases. Addition of 4-pentynoyl-coenzyme A, an alkynyl chemical reporter for protein acetylation, to cell extracts, together with purified lysine acetyltransferase p300, enabled the fluorescent profiling and identification of protein substrates via Cu(I)-catalyzed alkyne-azide cycloaddition. We identified several known protein substrates of the acetyltransferase p300 as well as the lysine residues that were modified. Interestingly, several new candidate p300 substrates and their sites of acetylation were also discovered using this approach. Our results demonstrate that bioorthogonal chemical proteomics allows the rapid substrate identification of individual protein acetyltransferases in vitro.
authors.affiliation: Pfizer, Pearl River, NY 10965, USA.
authors.forename: Yu-Ying
authors.initials: YY
authors.lastname: Yang
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu-Ying
authors.forename: Markus
authors.initials: M
authors.lastname: Grammel
authors.forename: Grammel
authors.initials: G
authors.lastname: Markus
authors.forename: Howard C
authors.initials: HC
authors.lastname: Hang
authors.forename: Hang C
authors.initials: HC
authors.lastname: Howard
mesh_terms: D000123:Acetyltransferases / Q000378:metabolism*; D002853:Chromatography, Liquid; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008239:Lysine / Q000378:metabolism*; D040901:Proteomics; D053719:Tandem Mass Spectrometry; D050880:p300-CBP Transcription Factors / Q000378:metabolism*; D000099224:p300-CBP-Associated Factor
chemical_list: D000123:Acetyltransferases; D003065:Coenzyme A; D008239:Lysine; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor