Fig. 3. Metabolite annotation from knowns to unknowns.

a Generation of unknown metabolites from a mixture of 46 metabolites (46std_mix) with in silico reactions or enzymatic biotransformation via human liver S9 fraction incubation. b, c Annotated peaks in positive (b) and negative ionization modes (c); the left cyclic bars represent the annotated peaks in different networks. The right bar represents the involved biotransformation in an unknown annotation. d–f Unknown annotations from seed metabolite inosine 5′-monophosphate (IMP, L0019): d IMP generates eight unknowns through four transformations in the knowledge-based metabolic reaction network; e knowledge-guided MS2 similarity network annotates two unknowns from the seed; f 29 different ion forms were annotated from three metabolites in global peak correlation network. g, h Validation of two annotated unknowns using chemical standards: inosine (g, labeled as Exd000182) and inosine 5′-sulfate (h, labeled as Exd006673). i Validation of annotated unknowns with different strategies. Source data are provided as a Source Data file.