| MS Description
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BioSource amount<br />
We weighed 70 … BioSource amount<br />
We weighed 70 mg dry weight (DW) of the lyophilized samples for CE-TOF-MS analysis, 5 mg DW for GC-TOF-MS analysis, 50 mg DW for LC-q-TOF-MS analysis to detect polar metabolites, and 15 mg DW for lipid profiling. <br />
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Extraction for CE-TOF-MS<br />
Seventy mg DW of each sample was extracted in 20 volumes of methanol containing 8 μM 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 15,000 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.<br />
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CE-TOF-MS conditions<br />
All CE-TOFMS experiments were performed using an Agilent G7100A CE Instrument (Agilent Technologies, Sacramento, CA), an Agilent G6224A TOF LC/MS system, an Agilent 1200 Infinity series G1311C Quad Pump VL, and the G1603A Agilent CE-MS adapter and G1607A Agilent CE-ESI-MS sprayer kit. The G1601BA 3D-CE ChemStation software for CE and G3335-64002 MH Workstation were used.<br />
Separation column and electrolytes: <br />
Separations were carried out using a fused silica capillary (50 μm i.d. × 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.<br />
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Sample injection: <br />
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.<br />
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Separation parameters: <br />
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.<br />
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Ionization: <br />
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.<br />
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Dry gas condition: <br />
A flow rate of heated dry nitrogen gas (heater temperature 300 °C) was maintained at 10 psig.<br />
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Voltage settings in TOF-MS: <br />
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.<br />
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Mass calibration: <br />
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.<br />
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Mass data acquirement: <br />
Exact mass data were acquired at a rate of 1.5 cycles/sec over a 50-1000 m/z range.<br />
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Quality control: <br />
In an every single sequence analysis (maximum 36 samples) on our CE-TOF-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.<br />
<br /> ompounds mixture.<br />
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