TY - JOUR
T1 - Pathway-specific metabolome analysis with 18 O 2 -labeled Medicago truncatula via a mass spectrometry-based approach
AU - Kera, Kota
AU - Fine, Dennis D.
AU - Wherritt, Daniel J.
AU - Nagashima, Yoshiki
AU - Shimada, Norimoto
AU - Ara, Takeshi
AU - Ogata, Yoshiyuki
AU - Sumner, Lloyd W.
AU - Suzuki, Hideyuki
N1 - Funding Information:
Funding This study was funded by Strategic International Collaborative Research Program (SICORP) of JST and NSF as shown in acknowledgement.
Funding Information:
Acknowledgements This work was supported by Strategic International Collaborative Research Program (SICORP), JST, and Japan Advanced Plant Science Network. We would also like to acknowledge support from the USA National Science Foundation Award #1139489 which was a joint initiative with the Japanese Science and Technology Agency Metabolomics for a Low Carbon Society program, USA NSF RCN Award#1340058, and USA NSF MRI #1126719. We would like to thank Dr. Nozomu Sakurai, Mr. Manabu Yamada, Dr. Kazuto Man-nen for supporting data analysis.
Publisher Copyright:
© 2018, The Author(s).
PY - 2018/5/1
Y1 - 2018/5/1
N2 - 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.
AB - 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.
KW - Flavonoid
KW - Medicago truncatula
KW - Metabolite modification
KW - Stable-isotope
KW - Untargeted metabolome analysis
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U2 - 10.1007/s11306-018-1364-6
DO - 10.1007/s11306-018-1364-6
M3 - Article
C2 - 29780292
AN - SCOPUS:85047016288
VL - 14
JO - Metabolomics
JF - Metabolomics
SN - 1573-3882
IS - 5
M1 - 71
ER -