FeatureMSEA: Metabolic Feature-based Metabolite Set Enrichment Analysis

Abstract Liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics detects thousands of metabolic features, but converting these chemical signals into metabolite set-level biological knowledge remains challenging. This is because most features lack unambiguous metabolite identities. Conventional metabolite set enrichment analysis (MSEA) generally requires identified metabolites and metabolite-level ranked inputs, leaving much of the untargeted feature space unused. Here, we present FeatureMSEA, a feature rank-based framework for metabolite set enrichment directly from metabolic features with ambiguous annotations. FeatureMSEA integrates multi-evidence feature-to-metabolite annotation, feature rank-based enrichment scoring, permutation-based inference, and iterative leading-edge-guided annotation refinement, with an optional LLM-assisted module for post-enrichment interpretation. In null comparisons of randomly split healthy samples, FeatureMSEA detected no significant metabolite sets, whereas metabolite-set spike-in simulations showed recovery of implanted signals. In a cerebrospinal fluid metabolomics study of Huntington’s disease, FeatureMSEA identified dysregulated metabolite sets related to amino acid metabolism, mitochondrial energy metabolism, and neuroactive signaling. MS/MS-based annotation analysis further showed that FeatureMSEA refinement reduced annotation ambiguity and prioritized chemically consistent candidate metabolites. In summary, FeatureMSEA provides a general framework for extracting metabolite set-level biological insights from LC-MS untargeted metabolomics in which confident metabolite identification remains incomplete.