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BioSource amount
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.
Extraction for CE-MS
50 mg of each sample was extracted in 20 volumes of methanol
containing 8µM of two reference compounds (methionine sulfone for cation and camphor 10-
sulfonic acid for anion analyses) using a Retsch mixer mill MM310 at a frequency of 27 Hz for
1 min. The extracts were then centrifuged at 20,400 x g for 3 min at 4 °C. Five hundred-µl
aliquot of the supernatant was transferred into a tube. Five hundred µl of chloroform and 200
µl of water was added into the tube to perform liquid-liquid distribution. The upper layer was
evaporated for 30 min at 45°C by a centrifugal concentrator to obtain two layers. For removing
high-molecular-weight compounds such as oligo-sugars, the upper layer was centrifugally filtered
through a Millipore 5-kDa cutoff filter at 9,100 g for 120 min at 4°C. The filtrate was dried for 120
min by a centrifugal concentrator. The residue (ca. 25 mg of each sample) was dissolved into 20
µl of water containing 200 µM of internal standards (3-aminopyrrolidine for cation and trimesic
acid for anion analyses) that were used for compensation of migration time in the peak annotation
step.
CE-TOF-MS conditions
All CE-TOFMS experiments were performed using an Agilent CE
capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany), an Agilent G3250AA
LC/MSD TOF system (Agilent Technologies, Palo Alto, CA), an Agilent 1100 series binary HPLC
pump, and the G1603A Agilent CE-MS adapter and G1607A Agilent CE-ESI-MS sprayer kit. The
G2201AA Agilent ChemStation software for CE and the Analyst QS software for TOFMS were
used.
Separation column and electrolytes:
Separations were carried out using a fused silica
capillary (50 µm i.d. x 100 cm total length) filled with 1 M formic acid for cation analyses or
with 20 mM ammonium formate (pH 10.0) for anion analyses as the electrolyte. The capillary
temperature was maintained at 20 °C.
Sample injection:
The sample solutions (11.25 µl of extracts, ca. 0.6 µg of each sample) were
injected at 50 mbar for 15 sec (15 nl). The sample tray was cooled below 4 °C.
Separation parameters:
Prior to each run the capillary was flushed with electrolyte for 5
min. The applied voltage for separation was set at 30 kV. Fifty percent (v/v) methanol/water
containing 0.5 µM reserpine was delivered as the sheath liquid at 10 µL/min.
Ionization:
ESI-TOFMS was conducted in the positive ion mode for cation analyses or in the
negative ion mode for anion analyses, and the capillary voltage was set at 4 kV.
Dry gas condition: A flow rate of heated dry nitrogen gas (heater temperature 300 °C) was
maintained at 10 psig.
Voltage settings in TOF-MS:
The fragmentor, skimmer, and Oct RFV voltage were set at
110V, 50V, and 160V for cation analyses or at 120V, 60V, and 220V for anion analyses, respectively.
Mass calibration:
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) for cation analyses or the formic acid dimer ion ([2M-H]-,
m/z = 91.0037) and reserpine ([M-H]−, m/z = 607.2661) for anion analyses provided the lock
mass for exact mass measurements.
Mass data acquirement:
Exact mass data were acquired at a rate of 1.5 cycles/sec over a 50-1000 m/z range.
Quality control:
In an every single sequence analysis (maximum 36 samples) on our CE-MS
system, we analyzed the standard compound mixture at the first and the end of sample analyses.
The detected peak area of standard compound mixture was checked in point of reproducible
sensitivity. Standard compound mixture composed of major detectable metabolites including
amino acids and organic acids, and this mixture was newly prepared at least once a half year.
In all analyses in this study, there were no differences in the sensitivity of standard compounds
mixture.
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