Lysine L-lactylation is the dominant lactylation isomer induced by glycolysis.

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.