Isotope-Coded Click Chemistry-Enabled Enrichment for Reliable Profiling and Quantification of Carboxyl Metabolites

Abstract Carboxyl-containing metabolites are involved in diverse biological processes and are closely associated with metabolic diseases. However, their sensitive and reliable profiling by liquid chromatography−mass spectrometry (LC−MS) remains challenging because of low endogenous abundance, inefficient electrospray ionization, and substantial interference from complex biological matrices. Here, we developed a click chemistry-enabled enrichment (CCEE) strategy that integrates isotope-coded derivatization with chemoselective enrichment for sensitive and selective profiling and quantification of carboxyl-containing metabolites in biological samples. CCEE markedly improved detection sensitivity and chromatographic performance while effectively reducing matrix interference. The method also exhibited good linearity, reproducibility, and quantitative accuracy. Application of CCEE to human serum enabled broad profiling of the carboxyl submetabolome and revealed metabolic alterations associated with metabolic dysfunction-associated fatty liver disease (MAFLD). Targeted absolute quantification of representative metabolites further validated the observed alterations. Notably, serum bilirubin levels were significantly lower in individuals with MAFLD and strongly correlated with multiple clinical indicators. Overall, CCEE provides a sensitive and selective strategy for profiling and quantifying the carboxyl submetabolome, facilitating the investigation of metabolic dysregulation and the discovery of candidate biomarkers for metabolic diseases.