Chemoselective Metabolomics via a Modular Reactivity‐Encoding Platform

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.