Chemoselective Metabolomics via a Modular Reactivity‐Encoding Platform

ABSTRACT Understanding disease‐associated metabolic reprogramming requires comprehensive interrogation of the chemically diverse metabolome. However, conventional liquid chromatography‐mass spectrometry (LC‐MS) workflows analyze metabolites in a largely non‐discriminatory manner, resulting in systematic underrepresentation of specific functional and reactivity classes due to heterogeneous ionization efficiencies and matrix interference. Here, we report a chemoselective metabolomics strategy based on a modular reactivity‐encoding platform (MREP) that enables functional group‐resolved stratification of complex metabolomes. Four orthogonally designed alkyne‐tagged probes selectively derivatize carboxyl, carbonyl, amine, and thiol functionalities under compatible conditions. The encoded metabolites are subsequently immobilized via azide‐alkyne cycloaddition onto a unified solid‐phase capture resin, which simultaneously removes matrix components and installs a diagnostic reporter module. This integrated encoding‐capture architecture achieves high reaction orthogonality, near‐quantitative conversion, and robust quantitative performance across structurally diverse metabolites. The resulting triazole derivatives exhibit markedly enhanced ionization efficiencies and generate a universal reporter‐ion, enabling confident submetabolome classification and reconstruction. Application to serum and liver tissues from mice substantially expands the detectable chemical space, yielding 7 208 features and 1 573 annotated metabolites across four functional group‐defined layers. Collectively, this work establishes the MREP framework as a versatile platform for reactivity‐resolved interrogation of complex small‐molecule systems.