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

ABSTRACT:

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 (¹⁸O₂) 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 ¹⁸O₂-metabolome analysis.

Methods

¹⁸O₂ labeled whole Medicago truncatula seedlings were prepared using ¹⁸O₂-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 ¹⁸O₂ 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 ¹⁸O₂. To the best of our knowledge, this is the first report of apigenin 4'-glucuronide in M. truncatula. Using MSⁿ analysis, we estimated that ¹⁸O atoms were specifically incorporated in apigenin, the coumaroyl group, and glucuronic acid. For apigenin, an ¹⁸O atom was incorporated in the 4'-hydroxy group. Thus, non-specific incorporation of an ¹⁸O atom by recycling during one month of labeling is unlikely compared with the more specific oxygenase-catalyzing reaction.

Conclusion

Our finding indicated that ¹⁸O₂ 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.

SUBMITTER: Kera K 

PROVIDER: S-EPMC5948250 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature