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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11306-018-1364-6) contains supplementary material, which is available to authorized users.