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Sixteen plants were planted on a single pl … Sixteen plants were planted on a single plate separated into fourths to minimize the differences in growth conditions. Four wild-type plants were planted on one-fourth, and four bsas mutant plants were planted on each of the remaining fourths. Five plates were replicated for each bsas mutant. Each sample was extracted with a concentration of 25 mg fresh weight of tissues per microliter of the extraction medium (methanol:chloroform:water [3:1:1; v/v/v]) by using a Retsh mixer mill MM 310 at a frequency of 30 Hz−1 for 3 min at 4°C. After centrifugation for 5 min at 15,100g, 400 μL of the supernatant of each plate were put together in accordance with each section of fourths. Four hundred microliters of the 2-mL supernatant were used for GC-TOF/MS analysis, and another 400 μL were used for CE-TOF/MS analysis.<br /><br />Analysis of metabolites by CE-TOF/MS was performed using an Agilent CE capillary electrophoresis system (Agilent Technologies), an Agilent G3250AA LC/MSD TOF system (Agilent Technologies), an Agilent 1100 series binary HPLC pump, and the Agilent G1603A CE-MS adapter and Agilent G1607A CE-ESI-MS sprayer kit. Agilent G2201AA ChemStation software for CE and Analyst QS software for TOF/MS were used. For cationic compounds, separations were carried out using a fused silica capillary (50 μm i.d. × 100 cm total length) filled with 1 m formic acid as the electrolyte. The sample solutions were injected at 50 mbar for 15 s (15 nL). Prior to each run, the capillary was flushed with electrolyte for 5 min. The applied voltage was set at 30 kV. The capillary temperature was maintained at 20°C, and the sample tray was cooled below 4°C. Fifty percent (v/v) methanol-water containing 0.5 μm reserpine was delivered as the sheath liquid at 10 μL min−1. ESI-TOF/MS was conducted in the positive ion mode and the capillary voltage was set at 4 kV. A flow rate of heated dry nitrogen gas (heater temperature 300°C) was maintained at 10 psig. In TOF/MS, the fragmentor, skimmer, and Oct RFV voltage were set at 110, 50, and 160 V, respectively. In acquiring a fragment ion mass spectrum, the fragmentor voltage was increased to 210 V. Automatic recalibration of each acquired spectrum was performed using reference masses of reference standards. The methanol dimer ion ([2M + H]+; m/z 65.0597) and reserpine ([M + H]+; m/z 609.2806) provided the lock mass for exact mass measurements. Exact mass data were acquired at a rate of 1.5 cycles s−1 over a 50 to 1,000 m/z range. Analysis of anionic compounds and nucleotides was carried out as described previously (Soga et al., 2002a, 2002b). ed previously (Soga et al., 2002a, 2002b).
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