Pathway-specific metabolome analysis with 18O2-labeled Medicago truncatula via a mass spectrometry-based approach

Oxygen from carbon dioxide, water or molecular oxygen, depending on the responsible enzyme, can lead to a large variety of metabolites through chemical modification. Pathway-specific labeling using isotopic molecular oxygen ( 18 O 2 ) makes it possible to determine the origin of oxygen atoms in metabolites and the presence of biosynthetic enzymes (e.g., oxygenases). In this study, we established the basis of 18 O 2 -metabolome analysis. 18 O 2 labeled whole Medicago truncatula seedlings were prepared using 18 O 2 -air and an economical sealed-glass bottle system. Metabolites were analyzed using high-accuracy and high-resolution mass spectrometry. Identification of the metabolite was confirmed by NMR following UHPLC–solid-phase extraction (SPE). A total of 511 peaks labeled by 18 O 2 from shoot and 343 peaks from root were annotated by untargeted metabolome analysis. Additionally, we identified a new flavonoid, apigenin 4′- O -[2′- O -coumaroyl-glucuronopyranosyl-(1–2)- O -glucuronopyranoside], that was labeled by 18 O 2 . To the best of our knowledge, this is the first report of apigenin 4′-glucuronide in M. truncatula . Using MS n analysis, we estimated that 18 O atoms were specifically incorporated in apigenin, the coumaroyl group, and glucuronic acid. For apigenin, an 18 O atom was incorporated in the 4′-hydroxy group. Thus, non-specific incorporation of an 18 O atom by recycling during one month of labeling is unlikely compared with the more specific oxygenase-catalyzing reaction. Our finding indicated that 18 O 2 labeling was effective not only for the mining of unknown metabolites which were biosynthesized by oxygenase-related pathway but also for the identification of metabolites whose oxygen atoms were derived from oxygenase activity.