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Plant and Cell Physiology Advance Access originally published online on December 2, 2008
Plant and Cell Physiology 2009 50(1):37-47; doi:10.1093/pcp/pcn183
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved.
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and the Japanese Society of Plant Physiologists are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Special Issue - Regular Paper

Widely Targeted Metabolomics Based on Large-Scale MS/MS Data for Elucidating Metabolite Accumulation Patterns in Plants

Yuji Sawada1,2, Kenji Akiyama1, Akane Sakata1, Ayuko Kuwahara1,2, Hitomi Otsuki1, Tetsuya Sakurai1, Kazuki Saito1,3 and Masami Yokota Hirai1,2,*

1RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045 Japan
2JST, CREST, 4-1-8 Hon-chou, Kawaguchi, Saitama, 332-0012 Japan
3Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba, Chiba, 263-8522 Japan

*Corresponding author: E-mail, myhirai{at}psc.riken.jp; Fax, +81-45-503-9491


  Abstract

Metabolomics is an ‘omics’ approach that aims to analyze all metabolites in a biological sample comprehensively. The detailed metabolite profiling of thousands of plant samples has great potential for directly elucidating plant metabolic processes. However, both a comprehensive analysis and a high throughput are difficult to achieve at the same time due to the wide diversity of metabolites in plants. Here, we have established a novel and practical metabolomics methodology for quantifying hundreds of targeted metabolites in a high-throughput manner. Multiple reaction monitoring (MRM) using tandem quadrupole mass spectrometry (TQMS), which monitors both the specific precursor ions and product ions of each metabolite, is a standard technique in targeted metabolomics, as it enables high sensitivity, reproducibility and a broad dynamic range. In this study, we optimized the MRM conditions for specific compounds by performing automated flow injection analyses with TQMS. Based on a total of 61,920 spectra for 860 authentic compounds, the MRM conditions of 497 compounds were successfully optimized. These were applied to high-throughput automated analysis of biological samples using TQMS coupled with ultra performance liquid chromatography (UPLC). By this analysis, approximately 100 metabolites were quantified in each of 14 plant accessions from Brassicaceae, Gramineae and Fabaceae. A hierarchical cluster analysis based on the metabolite accumulation patterns clearly showed differences among the plant families, and family-specific metabolites could be predicted using a batch-learning self-organizing map analysis. Thus, the automated widely targeted metabolomics approach established here should pave the way for large-scale metabolite profiling and comparative metabolomics.

Keywords: Automated - Comparative metabolomics - Liquid chromatography–mass spectrometry - Metabolite accumulation pattern - Multiple reaction monitoring - Targeted metabolomics

Abbreviations: ALHS, automated liquid handling system; APCI, atmospheric pressure chemical ionization; CE, collision energy; CV, cone voltage; EI, electron ionization; ESI, electrospray ionization; FIA, flow injection analysis; GC, gas chromatography; LC, liquid chromatography; MRM, multiple reaction monitoring; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MS2T, MS/MS spectral tag; NMR, nuclear magnetic resonance; Q, quadrupole; S/N ratio, signal-to-noise ratio; TOF, time-of-flight; TQMS, tandem quadrupole mass spectrometry; UPLC, ultra performance liquid chromatography.

(Received October 8, 2008; Accepted November 23, 2008)
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