Discovery of Novel Lactoyl-Metabolites by Integrating Chemical Isotope Labeling and Knowledge-Based Prediction

Lactylation is a widespread protein modification with important regulatory roles in health and disease. Emerging evidence also links metabolite lactylation to disease onset, progression, and treatment response. It is estimated that an uncharacterized pool of lactoyl-metabolites exists in vivo, according to its formation mechanism. To mine this pool, we combined knowledge-driven prediction with stable isotopic labeling-based high-resolution mass spectrometry (SIL-HRMS). A predictive library of 151 lactoyl-metabolites was generated by expanding 20 basic amino acids (AAs) to their upstream and downstream metabolites. To improve detection, we developed a pair of new hydroxyl-reactive labeling reagents, 3,5-(dimethylamino)-2,4,6-triazine benzene-1-chlorine (Tmt-aycl-Cl) and its deuterium form d₁₂-Tmt-aycl-Cl. Using HRMS, 95 lactoyl-metabolites were putatively identified across 14 tissue types, 64 structures of which are reported for the first time. Representative lactoyl-metabolites were chemically synthesized and characterized to verify some of the annotations. We then developed a pseudotargeted metabolomics workflow for semiquantitative profiling in a diabetic kidney disease (DKD) mouse model. DKD mice showed elevated lactate in serum and the kidney and widespread dysregulations of lactoyl-metabolites. Many lactoyl-metabolites correlated strongly with the urinary albumin-to-creatinine ratio, suggesting biomarker potential for DKD. This integrated strategy expands the catalog of lactoyl-metabolites and provides a platform for investigating their biological significance.