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

Kota Kera, Dennis D. Fine, Daniel J. Wherritt, Yoshiki Nagashima, Norimoto Shimada, Takeshi Ara, Yoshiyuki Ogata, Lloyd W. Sumner, Hideyuki Suzuki

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)


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.

Original languageEnglish
Article number71
Issue number5
Publication statusPublished - 2018 May 1


  • Flavonoid
  • Medicago truncatula
  • Metabolite modification
  • Stable-isotope
  • Untargeted metabolome analysis

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Clinical Biochemistry


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