Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge CB2 0XY, UK.
School of Chemistry, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
Cell Chem Biol. 2022 Jul 21;29(7):1232-1244.e5. doi: 10.1016/j.chembiol.2022.04.005.
During metabolism, carboxylic acids are often activated by conjugation to the thiol of coenzyme A (CoA). The resulting acyl-CoAs comprise a group of ∼100 thioester-containing metabolites that could modify protein behavior through non-enzymatic N-acylation of lysine residues. However, the importance of many potential acyl modifications remains unclear because antibody-based methods to detect them are unavailable and the in vivo concentrations of their respective acyl-CoAs are poorly characterized. Here, we develop cysteine-triphenylphosphonium (CysTPP), a mass spectrometry probe that uses "native chemical ligation" to sensitively detect the major acyl-CoAs present in vivo through irreversible modification of its amine via a thioester intermediate. Using CysTPP, we show that longer-chain (C13-C22) acyl-CoAs often constitute ∼60% of the acyl-CoA pool in rat tissues. These hydrophobic longer-chain fatty acyl-CoAs have the potential to non-enzymatically modify protein residues.
在新陈代谢过程中，羧酸通常通过与辅酶 A（CoA）的巯基结合而被激活。由此产生的酰基辅酶 A 包含一组约 100 种含有硫酯的代谢物，这些代谢物可以通过赖氨酸残基的非酶促 N-酰化来改变蛋白质的行为。然而，许多潜在酰基修饰的重要性仍然不清楚，因为缺乏基于抗体的检测方法，并且它们各自的酰基辅酶 A 的体内浓度特征描述较差。在这里，我们开发了半胱氨酸-三苯基膦（CysTPP），这是一种质谱探针，通过“天然化学连接”使用硫酯中间体不可逆修饰其胺基来灵敏地检测体内存在的主要酰基辅酶 A。使用 CysTPP，我们表明长链（C13-C22）酰基辅酶 A 通常构成大鼠组织中酰基辅酶 A 池的约 60%。这些疏水性长链脂肪酸酰基辅酶 A 有可能非酶促修饰蛋白质残基。
