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Metabolite pull-down is an affinity-based chemical proteomics technique in which a target metabolite (or its biotin/photoaffinity probe) is immobilized on a solid-phase matrix and incubated with cell lysates or protein mixtures to selectively capture its direct protein binding partners. After stringent washing to remove non-specific interactions, the enriched proteins are identified by mass spectrometry (MS), thereby elucidating the molecular mechanism of action of the metabolite. This approach is widely used in drug target discovery, natural product mechanism deconvolution, and signaling pathway investigation.