Large-Scale Metabolite Annotation in Untargeted Metabolomics Using MetDNA

MetDNA ( http://metdna.zhulab.cn/ ) is a network-based computational platform for large-scale metabolite annotation in untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS). By using a metabolic reaction network (MRN) to guide recursive MS2 spectral similarity matching, MetDNA can accurately annotate both known and unknown metabolites, going beyond the limits of conventional spectral libraries. Since 2019, the platform has evolved from MetDNA to MetDNA2 and now to MetDNA3, with improvements in efficiency, coverage, and confidence in metabolite annotation for untargeted metabolomics. In this protocol, we outline best practices for data preparation, parameter configuration, and result interpretation, offering users a practical workflow to maximize the utility of MetDNA for high-confidence metabolite annotation.