GC-EI-TOF-MS analysis of in vivo carbon-partitioning into soluble metabolite pools of higher plants by monitoring isotope dilution after ¹³CO₂ labelling

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• 作者：Jan Huege ; Ronan Sulpice ; Yves Gibon ; Jan Lisec ; Karin Koehl ; Joachim Kopka
• 关键词：Arabidopsis thaliana Col-0 ; Oryza sativa IR57111 ; ¹³C-carbon ; ¹³CO₂-carbondioxide ; Dynamic flux analysis ; Electron impact ionization (EI) ; Gas chromatography (GC) ; Metabolite profiling ; Stable isotope dilutio
• 刊名：Phytochemistry
• 出版年：2007
• 期刊代码：85_00319422
• 类别：ch
• 出版时间：August-September 2007
• 卷：68
• 期：16-18
• 页码：2258-2272
• 文件大小：357 K

摘要

The established GC-EI-TOF-MS method for the profiling of soluble polar metabolites from plant tissue was employed for the kinetic metabolic phenotyping of higher plants. Approximately 100 typical GC-EI-MS mass fragments of trimethylsilylated and methoxyaminated metabolite derivatives were structurally interpreted for mass isotopomer analysis, thus enabling the kinetic study of identified metabolites as well as the so-called functional group monitoring of yet non-identified metabolites. The monitoring of isotope dilution after ¹³CO₂ labelling was optimized using Arabidopsis thaliana Col-0 or Oryza sativa IR57111 plants, which were maximally labelled with ¹³C. Carbon isotope dilution was evaluated for short (2 h) and long-term (3 days) kinetic measurements of metabolite pools in root and shoots. Both approaches were shown to enable the characterization of metabolite specific partitioning processes and kinetics. Simplifying data reduction schemes comprising calculation of ¹³C-enrichment from mass isotopomer distributions and of initial ¹³C-dilution rates were employed. Metabolites exhibited a highly diverse range of metabolite and organ specific half-life of ¹³C-label in their respective pools (¹³C-half-life). This observation implied the setting of metabolite specific periods for optimal kinetic monitoring. A current experimental design for the kinetic metabolic phenotyping of higher plants is proposed.