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

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.

Primary Metabolism during Biosynthesis of Secondary Wall Polymers of Protoxylem Vessel Elements

“…After harvesting, the cells were freeze-dried in a vacuum freeze dryer (FZ-2.5; Labconco) and then subjected to wide-target metabolome analysis by the methods described by Sawada et al (2009) with minor modifications. The freeze-dried BY-2 cell samples were crushed with a bead shaker (1,000 rpm, 2 min; ShakeMaster Neo; BMS), and 4 6 0.2 mg of sample was extracted with 80% (v/v) methanol by crushing with 5-mm zirconia beads (1,000 rpm for 2 min).…”

“…Induced BY-2 cells were subjected to liquid chromatography-mass spectrometry (LC-MS)-based wide-target metabolome analysis as established by Sawada et al (2009), which can automatically detect approximately 700 metabolites that are the products of metabolic regulatory pathways (Sawada et al, 2009). This method can provide data on general metabolites during protoxylem vessel element formation with high reproducibility.…”

“…Using the LC-MS-based wide-target metabolome analysis established by Sawada et al (2009), we identified 490 metabolites from the control and VND7-VP16-GR BY-2 cells and obtained relative amount data for 128 metabolites based on a cutoff signal-to-noise ratio of greater than 3 (Supplemental Tables S1 and S2). Principal component analysis (PCA) of these 128 metabolites successfully identified changes in the metabolome associated with the progression of cell differentiation; DEX-treated VND7-VP16-GR samples, mock-treated VND7-VP16-GR samples, and nontransgenic samples were separated in the PCA plot (Fig.…”

Nutritionally Enhanced Staple Food Crops

“…The newly developed methods of screening for potential alterations in the metabolism of the modified organism include the analysis of gene expression (microarrays, mRNA finger printing), overall protein analysis (proteomics), and secondary metabolites (HT-UHPLS-TOF-MS, MRM-TQMS; Grata et al 2009;Sawada et al 2009;Allwood and Goodacre 2010) that should be integrated into the risk assessment process. To sum up, the advances made in molecular biology, toxicology, biochemistry, and nutrition hold the promise of providing sets of genes and methodologies that serve as biomarkers for a cell's responses to toxins, allergens, or other compounds, which hold potential to the development of new tools to assess the GM crops.…”

Technical Challenges in Mass Spectrometry-Based Metabolomics

“…37) Sawada et al reported widely targeted metabolome analysis using liquid chromatographtriple quadrupole-mass spectrometers (TQ-MS) by developing selected ion monitoring series of 378 metabolites. 38) Similar approaches are employed for the comprehensive analysis of lipids using liquid chromatograph-TQ-MS (lipidomics) 39) and analysis of primary metabolites using gas chromatograph-TQ-MS. 40) Although the number of measurable metabolites depends on the samples of interests, it has been reported that approximately 100-200 metabolites were usually analyzed by the widely targeted analysis. e widely targeted approach is now a productive and realistic strategy in metabolomics because the analysis could be performed using the more popular TQ-MS and default peak picking so ware developed by manufacturers.…”