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<Automated liquid handling for sample p … <Automated liquid handling for sample preparation><br />
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).
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<Acquisition of ESI-MS and ESI-MS/MS data><br />
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).<br /><br />
<UPLC-TQMS analysis><br />
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.<br /> source temperature was 130°C.<br />
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