| MS Description
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BioSource amount<br />
For RDRS and … BioSource amount<br />
For RDRS and Hoshiyutaka, 100 seeds of each variety were selected according to the average weight
and length of seeds. After separating the husks from the seeds, the brown rice seeds obtained were
bulked and crushed by using a Retsch mixer mill MM301 at a frequency of 20 Hz for 2 min at
4 °C. Successively, the obtained powder was divided into three to four pools. For external set of
samples harvested in Akita, 100 seeds of each biological replicate were selected and crushed in the
same way as RDRS.<br /><br />
Extraction for IT-MS<br />
Each Sample (50 mg) was extracted with 750 µl of chloroform/MeOH
(1:1, v/v) containing 1.25 µM 1,2-dioctanoyl-sn-glycero-3-phosphocholine (SIGMA) followed by
centrifugation at 10,000g at 4°C for 5 min. The supernatant was transferred to a 2 ml tube,
and the extraction procedure was repeated again. The combined supernatant was evaporated to
dryness by SPD2010 SpeedVac® concentrator. The residue was dissolved in 750 µl of ethanol,
and centrifuged at 10,000g at 4°C for 5 min. Six hundred microlitter of the supernatant was
transferred to a glass tube for polar-lipid analysis.
<br /><br />
LC-IT-TOF-MS conditions<br />
Extracts (0.5 µl, ca. 33.3 µg of each sample) was analyzed by LCMS
with ESI interface (LC, Shimadzu LC-20AD system; MS, Shimadzu LCMS-IT-TOF) operated
by Shimadzu LCMSsolution software (version 3.60). Two-solvent system was used for separation of
each metabolite. The analytical conditions were as follows. Column, Shim-pack XR-ODS (2.0 mm
I.D., 50 mm long); solvent A, water (1% 1M ammonium formate and 0.1% formic acid); solvent
B, acetonitrile/isopropyl alcohol (40:60, v/v. 1% 1M ammonium formate and 0.1% formic acid);
gradient program, 40% B at 0 min, 75% B at 3 min, 95% B at 10 min, 100% B at 19 min, 100% B at
27 min, 40% B at 27.01 min (total run time, 30 min); flow rate, 0.3 ml/min; column temperature,
55°C; MS interface voltage, 4.50 kV, nebulizer gas, 1.50 L/min; CDL temperature 200.0°C, heat
block temperature, 200°C; detection mode, scan (m/z 150˜1600, positive); scan time, 0.25 sec; ion
accumulation time, 20 msec. 0.25 sec; ion
accumulation time, 20 msec.
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