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.