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Tissue samples were cut into small pieces … Tissue samples were cut into small pieces and extracted with 40 volumes (w/v) of extraction solvent (methanol : water (50 : 50, v/v) containing 0.2% acetic acid) at room temperature for one hour. Extracts (500 ml) were added to 2.0 ml dilution solvent (methanol : water (90 : 10, v/v) containing 0.2% acetic acid) and applied to a Sep-Pak Plus C18 Cartridge (Waters, Milford, USA) equilibrated with dilution solvent. The cartridge was washed with 2.0 ml dilution solvent and all solvent in the cartridge was eluted by aeration. The eluate was evaporated under a vacuum and the residue was dissolved in 200 ml of 20% methanol in water. Following centrifugation at 15,000 g for 10 min, supernatants were subjected to LC-ESI-Q-MS analyses. Sample extracts (5 ml) were analyzed using a LC-MS system equipped with an electrospray ionization (ESI) interface (HPLC: Shimadzu LC-10VP system, MS; Shimadzu LCMS2010).
The analytical conditions were as follows: HPLC 18 Metabolic profiling of Trp overproducing rice Copyright © 2010 The Japanese Society for Plant Cell and Molecular Biology
column: Cadenza CD-C18, Imtact Co., Kyoto, Japan, 2.075 mm; solvent system: acetonitrile (0.1% formic acid) : water (0.1% formic acid), gradient program: 5:95, v/v, at 0 min; 60:40 at 11 min; 98:2 at 12 min; 98:2 at 13 min; 5:95 at 13.1 min; 5:95 at 20 min, flow rate: 0.25 ml min-1, temperature: 35°C, MS detection; CDL temperature: 250°C, block heater temperature: 200°C, probe voltage: +4.5 kV, Q-array voltage: scanning mode, nebulizing gas flow: 1.5 l min-1, detection mode: scan mode (m/z 100–700), scan time: 2 sec. The scans were repeated for 15 min (750 times) in a single run. Data were recorded with the aid of LCMS-Solution version 2.0 software (Shimadzu, Kyoto). on version 2.0 software (Shimadzu, Kyoto).
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