Pathway-specific metabolome analysis with ¹⁸ O ₂ -labeled Medicago truncatula via a mass spectrometry-based approach

Introduction: Oxygen from carbon dioxide, water or molecular oxygen, depending on the responsible enzyme, can lead to a large variety of metabolites through chemical modification. Objectives: 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. Methods: 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). Results: 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. Conclusion: 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.