SE57:/MS01
Sample Set Information
| ID | SE57 |
|---|---|
| Title | Widely targeted metabolomics based on large-scale MS/MS data for elucidating metabolite accumulation patterns in plants |
| Description | 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 quantifi ed 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-specifi c 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 profi ling and comparative metabolomics. |
| Authors | Yuji Sawada, Kenji Akiyama, Akane Sakata, Ayuko Kuwahara, Hitomi Otsuki, Tetsuya Sakurai, Kazuki Saito, Masami Yokota Hirai |
| Reference | Sawada Y et al. (2009) Plant and Cell Physiology 50: 37-47 |
| Comment |
The raw data files are available at DROP Met web site in PRIMe database of RIKEN.
Analytical Method Details Information
| ID | MS01 |
|---|---|
| Title | Acquisition of ESI-MS and ESI-MS/MS data, UPLC-TQMS analysis |
| Instrument | Waters Xevo TQD, UPLC (Waters) |
| Instrument Type | UPLC-QTOF-MS |
| Ionization | ESI |
| Ion Mode | Positive and Negative |
| Description | <Automated liquid handling for sample preparation> The plant tissue samples were frozen in liquid nitrogen, quenched using a Mixer Mill (MM300, Retsch, Hann, Germany) as previously reported ( Hirai et al. 2007 ), and the extraction buffer (80% MeOH) was added. The resulting extracts consisted of 400 μ l with 20 mg ml –1 (fresh weight) of tissue. The extracts were transferred and treated using an ALHS as follows (Supplementary Fig. S1): 350 μ l of each plant extract were transferred to a 96-well plate, and the solutions were dried under N 2 gas using a 96-well format spray instrument (40°C, 25 min and 30°C, 20 min). The dried samples were dissolved in 350 μ l of H 2 O using a vortex system (1,300 r.p.m., 6 min), and then fi ltered through a 96-well filter [Captiva 96-well Filter Plate (pore size 0.45 μ m, polyvinylidene fl uoride), Varian, CA, USA], using a vacuum manifold with the following program: 30 s, 50 Hpa; 20 s, 100 Hpa; 20 s, 200 Hpa; 30 s, 300 Hpa; and 60 s, 300 Hpa. Plate handling was carried out automatically using robot arms (iSWAP, Hamilton).
All the solutions of authentic compounds (250 μ M) were transferred from 10 ml vials to 1.2 ml glass inserts in 96-well plates (Webseal system, GL Sciences, Tokyo, Japan) and diluted to 50 pmol μ l –1 with H 2 O using an ALHS (Microlab STARplus, Hamilton, Reno, NV, USA). The solutions (20 μ l aliquots containing 1 nmol of compound) were analyzed by the flow injection method using a CTC-PAL injection system (AMR, Tokyo, Japan) and a Waters GI Pump solvent system (Waters, Milford, MA, USA) consisting of: 0.05 ml min –1 flow using a micro splitter (GL Sciences), 80% acetonitrile (0.1% formic acid) and 20% water (0.1% formic acid), with a 2.5 min cycle in the isocratic mode. The ionized authentic compounds were detected by TQMS (TQD, Waters) according to the following conditions: capillary voltage +3.0 keV or –2.8 keV, CV 10–60 eV (six levels), source temperature 120°C, desolvation temperature 350°C, cone gas fl ow 50 l h –1 , desolvation gas fl ow 600 l h –1 and CE 10–60 eV (six levels). A total of 61,920 spectra of 860 compounds were obtained.The optimal MRM conditions, including positive/negative polarity (e.g. [M] + , [M + H] + , [M] – , [M – H] – ), m / z of precursor ion and product ion, and optimal CV and CE were determined automatically for 497 of these compounds using the software QuanOptimize (Waters).
The UPLC (Waters) conditions were manually optimized based on the separation patterns of 12 methionine-derived glucosinolates and were as follows: flow rate 0.24 ml min –1 ; solvents A, 0.1% formic acid in water and B, 0.1% formic acid in acetonitrile; gradient program of B (0 min, 0%; 0.25 min, 0%; 0.4 min, 9%; 0.8 min, 17%; 1.9 min, 100%; 2.1 min, 100%;2.11 min, 0%); 3 min cycles with a temperature of 38°C.The TQMS detection conditions were the same as those for FIA-TQMS, except that the source temperature was 130°C. |
| Comment_of_details |
