Lysine l-lactylation (K_l-la) is a novel protein posttranslational modification (PTM) driven by l-lactate. This PTM has three isomers: K_l-la, N-ε-(carboxyethyl)-lysine (K_ce) and d-lactyl-lysine (K_d-la), which are often confused in the context of the Warburg effect and nuclear presence. Here we introduce two methods to differentiate these isomers: a chemical derivatization and high-performance liquid chromatography analysis for efficient separation, and isomer-specific antibodies for high-selectivity identification. We demonstrated that K_l-la is the primary lactylation isomer on histones and dynamically regulated by glycolysis, not K_d-la or K_ce, which are observed when the glyoxalase system was incomplete. The study also reveals that lactyl-coenzyme A, a precursor in l-lactylation, correlates positively with K_l-la levels. This work not only provides a methodology for distinguishing other PTM isomers, but also highlights K_l-la as the primary responder to glycolysis and the Warburg effect.

赖氨酸 l-乳酸化 （K_l-la） 是一种由 l-乳酸驱动的新型蛋白质翻译后修饰 （PTM）。这种 PTM 有三种异构体：K_l-la、N-ε-（羧乙基）-赖氨酸 （K_ce） 和 d-乳酰-赖氨酸 （K_d-la），它们在 Warburg 效应和核存在的背景下经常被混淆。本文介绍了两种区分这些异构体的方法：用于高效分离的化学衍生化和高效液相色谱分析，以及用于高选择性鉴定的异构体特异性抗体。我们证明 K_l-la 是组蛋白上的主要乳酰化异构体，并受糖酵解动态调节，而不是 K_d-la 或 K_ce，当乙二醛酶系统不完整时观察到。该研究还显示，乳酰辅酶 A 是 l-乳酰化的前体，与 _K-la 水平呈正相关。这项工作不仅提供了一种区分其他 PTM 异构体的方法，还强调了 K_l-la 是糖酵解和 Warburg 效应的主要反应者。