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J Am Chem Soc. 2010 Mar 24;132(11):3640-1. doi: 10.1021/ja908871t.

Bioorthogonal chemical reporters for monitoring protein acetylation.

Author information

1
Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York 10065, USA.

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.

PMID:
20192265
PMCID:
PMC2848987
DOI:
10.1021/ja908871t
[Indexed for MEDLINE]
Free PMC Article

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