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.

Figure 1

(a) Two-step labeling strategy for detecting protein acetylation in vitro and in cells. (b) In-gel fluorescent detection of p300-catalyzed histone H3 acylation. Coomassie blue (CB) gel shows protein loading control.

Figure 2

Fluorescent detection of acetate reporter (4, 5 and 6)(labeled (a) core histones, (b) purified histone H3 and (c) total cell lysates from Jurkat T cells.