Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York 10065, USA.
J Am Chem Soc. 2010 Mar 24;132(11):3640-1. doi: 10.1021/ja908871t.
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.
蛋白质乙酰化是一种关键的翻译后修饰，可调节真核生物中多种生物活性。在此，我们报告了生物正交化学报告物，可通过 Cu(I) 催化的叠氮-炔环加成（通常称为“点击化学”）直接在凝胶中进行荧光可视化和蛋白质组范围内鉴定乙酰化蛋白质。我们证明，两种炔基-乙酰辅酶 A 类似物，4-戊炔酰辅酶 A 和 5-己炔酰辅酶 A，可作为赖氨酸乙酰转移酶 p300 的有效底物，并可作为体外监测 p300 催化的蛋白质乙酰化的灵敏试剂。此外，我们证明，三种炔基乙酸盐类似物，即 3-丁炔酸钠、4-戊炔酸钠和 5-己炔酸钠，可通过生物合成机制代谢掺入到细胞蛋白质中，用于分析各种细胞类型中的乙酰化蛋白质。对富集的 4-戊炔酰化标记蛋白进行质谱分析，揭示了 Jurkat T 细胞中的许多已报道和新的候选乙酰化蛋白以及赖氨酸乙酰化的特定位点。这里描述的生物正交化学报告物应该成为研究蛋白质乙酰化的有力工具。
