8 安定同位体標識および多次元NMR法による、代謝物一斉解析法の開発と植物代謝動態解析の検討(口頭発表の部)

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The interdisciplinary field of systems biology has evolved rapidly in recent years. A prerequisite for deriving the benefits of such a systems approach is a reliable and well validated bioanalytical platform across complementary measurement modalities, especially transcriptomics, proteomics, and metabolomics. Of these, metabolome analysis is more difficult than genome or proteome analysis because of the diverse chemical and physical properties of metabolites. Nuclear magnetic resonance (NMR) techniques can provide information about the global pool of metabolites, including both soluble low-molecular-weight compounds and insoluble macromolecules. We have been developing new methodologies for metabolomics that combine stable isotope labeling and multidimensional heteronuclear NMR analysis. At this conference, we focus on the methodologies for measuring metabolites. To show the validity of our method, we also describe how we can obtain information on metabolic networks in ^<13>C-labeled Arabidopsis thaliana. The problem of spectral overlap in NMR spectroscopy encountered in NMR-based metabolomics can be alleviated by increasing the spectral dimensionality. However, a large amount of macromolecules, such as proteins, increases the line width and decreases the efficiency of magnetization transfer by spin-spin relaxation resulting in a decrease in detectable correlation signals. In order to establish extraction methodologies that are applicable to a wide range of organisms, we evaluated a series of NMR solvents using the distribution of the full-width at half-maximum and the number of signals observed in ^1H-^<13>C heteronuclear single quantum coherence (HSQC) spectra using ^<13>C-labeled plants, bacteria, and animal samples. In addition, we optimized the concentration of HEPES-d_<18> buffer to keep the pH and chemical shifts constant, and to avoid decreasing the sensitivity of the cryogenic probe. As a result, we chose CD_3OD/HEPES-d_<18> and hexafluoroacetone trideuterate (HFA・3D_2O)/HEPES-d_<18> buffer as extraction solvents. Detailed assignments of the metabolites will be presented. Furthermore, we examined the insoluble fractions, which are required to more fully appreciate all of the functions of metabolites and metabolic networks in whole cells, using high-resolution magic angle spinning (HR-MAS) analysis. Concerning metabolic network analysis, we investigated how to obtain information about biochemical reactions, C-C bond formation, and the cleavage of the major metabolites, such as free amino acids, in crude extracts based on the analysis of the ^<13>C-^<13>C coupling pattern in two-dimensional (2-D)-NMR spectra. For example, the combination of different extraction solvents allows one to distinguish complicated ^<13>C-^<13>C fine couplings at the Cα position of amino acids. As another approach, the f1-f3 projection of the HCACO spectrum also helps in the analysis of ^<13>C-^<13>C connectivities. Using these new methods, we present an example that involves monitoring the incorporation profile of [^<13>C_6]glucose into A. thaliana and its metabolic dynamics, which change in a time-dependent manner with atmospheric ^<12>CO_2 assimilation.
2007-08-24

8 安定同位体標識および多次元NMR法による、代謝物一斉解析法の開発と植物代謝動態解析の検討(口頭発表の部)

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