Systematic NMR Analysis of Stable Isotope Labeled Metabolite Mixtures in Plant and Animal Systems: Coarse Grained Views of Metabolic Pathways

Abstract

BackgroundMetabolic phenotyping has become an important ‘bird's-eye-view’ technology which can be applied to higher organisms, such as model plant and animal systems in the post-genomics and proteomics era. Although genotyping technology has expanded greatly over the past decade, metabolic phenotyping has languished due to the difficulty of ‘top-down’ chemical analyses. Here, we describe a systematic NMR methodology for stable isotope-labeling and analysis of metabolite mixtures in plant and animal systems.Methodology/Principal FindingsThe analysis method includes a stable isotope labeling technique for use in living organisms; a systematic method for simultaneously identifying a large number of metabolites by using a newly developed HSQC-based metabolite chemical shift database combined with heteronuclear multidimensional NMR spectroscopy; Principal Components Analysis; and a visualization method using a coarse-grained overview of the metabolic system. The database contains more than 1000 1H and 13C chemical shifts corresponding to 142 metabolites measured under identical physicochemical conditions. Using the stable isotope labeling technique in Arabidopsis T87 cultured cells and Bombyx mori, we systematically detected >450 HSQC peaks in each 13C-HSQC spectrum derived from model plant, Arabidopsis T87 cultured cells and the invertebrate animal model Bombyx mori. Furthermore, for the first time, efficient 13C labeling has allowed reliable signal assignment using analytical separation techniques such as 3D HCCH-COSY spectra in higher organism extracts.Conclusions/SignificanceOverall physiological changes could be detected and categorized in relation to a critical developmental phase change in B. mori by coarse-grained representations in which the organization of metabolic pathways related to a specific developmental phase was visualized on the basis of constituent changes of 56 identified metabolites. Based on the observed intensities of 13C atoms of given metabolites on development-dependent changes in the 56 identified 13C-HSQC signals, we have determined the changes in metabolic networks that are associated with energy and nitrogen metabolism.

Solid-, Solution-, and Gas-state NMR Monitoring of 13C-Cellulose Degradation in an Anaerobic Microbial Ecosystem

“…The acquired spectra were manually phased and baseline-corrected. NMR spectra were processed using NMRPipe software [34] and assigned using the SpinAssign program on the PRIMe website [35][36][37].…”

Non invasive analysis of metabolic changes following nutrient input into diverse fish species, as investigated by metabolic and microbial profiling approaches

“…The HSQC TOCSY spectra were acquired as 1024 (F2) and 256 (F1) data points with 180 scans and an interscan delay of 1 s with 16 dummy scans. The NMR spectra were processed using Bruker TopSpin software (Bruker BioSpin GmbH) and assigned using the SpinAssign program at the PRIMe website (http://prime.psc.riken.jp/) (Akiyama et al 2008;Chikayama et al 2010;Chikayama et al 2008) and the Biological Magnetic Resonance Bank (http://www.bmrb.wisc.edu/metabolomics/query_metab.php) (Ulrich et al 2008). …”

Chemical profiling of complex biochemical mixtures from various seaweeds

“…23,24 Peak assignments were made using the SpinAssign program on the PRIMe website (http://prime.psc.riken.jp/). [25][26][27] To evaluate the water-insoluble seaweed components, lyophilized samples were powdered and processed by various procedures to identify the best extraction method (Supplementary Table 2), and the optimal-modified process was used for all further analyses. In the optimal process, the lyophilized samples were roughly crushed in a food blender and then powdered using an Automill machine.…”

“…The characterization of the water-soluble components via an NMR-based metabolomics approach has been used extensively to study metabolites in a wide range of biological systems in various environments, [6][7][8][9][10][11][12][13] and is capable of creating assignments using our database (that is, SpinAssign program at the PRIMe website). [25][26][27] The water-insoluble …”