Lysine lactoylation is a recently described protein post-translational modification (PTM). However, the biochemical pathways responsible for this acylation remain unclear. Two metabolite-dependent mechanisms have been proposed: enzymatic histone lysine lactoylation derived from lactoyl-coenzyme A (lactoyl-CoA, also termed lactyl-CoA), and non-enzymatic lysine lactoylation resulting from acyl-transfer via lactoyl-glutathione. While the former has precedent in the form of enzyme-catalysed lysine acylation, the lactoyl-CoA metabolite has not been previously quantified in mammalian systems. Here, we use liquid chromatography–high-resolution mass spectrometry (LC-HRMS) together with a synthetic standard to detect and validate the presence of lactoyl-CoA in cell and tissue samples. Conducting a retrospective analysis of data from previously analysed samples revealed the presence of lactoyl-CoA in diverse cell and tissue contexts. In addition, we describe a biosynthetic route to generate ¹³C₃¹⁵N₁-isotopically labelled lactoyl-CoA, providing a co-eluting internal standard for analysis of this metabolite. We estimate lactoyl-CoA concentrations of 1.14 × 10⁻⁸ pmol per cell in cell culture and 0.0172 pmol mg⁻¹ tissue wet weight in mouse heart. These levels are similar to crotonyl-CoA, but between 20 and 350 times lower than predominant acyl-CoAs such as acetyl-, propionyl- and succinyl-CoA. Overall our studies provide the first quantitative measurements of lactoyl-CoA in metazoans, and provide a methodological foundation for the interrogation of this novel metabolite in biology and disease.

赖氨酸乳酰化是最近描述的一种蛋白质翻译后修饰（PTM）。然而，导致这种酰化的生化途径仍不清楚。已经提出了两种代谢物依赖性机制：源自乳酰辅酶 A（乳酰辅酶 A，也称为乳酰辅酶 A）的酶促组蛋白赖氨酸乳酰化，以及通过乳酰谷胱甘肽进行酰基转移产生的非酶促赖氨酸乳酰化。虽然前者以酶催化赖氨酸酰化的形式存在先例，但乳酰辅酶A代谢物此前尚未在哺乳动物系统中进行定量。在这里，我们使用液相色谱-高分辨率质谱 (LC-HRMS) 和合成标准品来检测和验证细胞和组织样品中乳酰辅酶 A 的存在。对先前分析的样本数据进行回顾性分析揭示了乳酰辅酶A在不同的细胞和组织环境中的存在。此外，我们描述了生成¹³ C ₃ ¹⁵ N ₁ -同位素标记的乳酰辅酶A的生物合成途径，为分析该代谢物提供了共洗脱内标。我们估计细胞培养物中每个细胞的乳酰辅酶A浓度为1.14 × 10 ^-8 pmol，小鼠心脏组织湿重为0.0172 pmol mg ^{-1 。}这些水平与巴豆酰辅酶A相似，但比主要酰基辅酶A（如乙酰辅酶A、丙酰辅酶A和琥珀酰辅酶A）低20至350倍。总的来说，我们的研究首次定量测量了后生动物中的乳酰辅酶A，并为研究这种新型代谢物在生物学和疾病中的应用提供了方法学基础。