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Sample metabolomic data were acquired usin … Sample metabolomic data were acquired using four time-of-flight mass spectrometers (TOF-MS), including CE-TOF-MS for analysis of polar metabolites, GC-TOF-MS for analysis of primary metabolites, LC-Q-TOF-MS for analysis of secondary metabolites and LC-IT-TOF-MS for analysis of lipids. Analysis was performed in triplicate, and the samples were extracted from each pipeline using optimized methods. <br />
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For the LC-Q-TOF-MS pipeline, 100 mg of rice seed powder was homogenized in 10 volumes of 5% aqueous methanol containing 0.1% acetic acid (Tokyo Kasei, http://www.tciamerica.com/) using an MM300 mixer mill (Retsch, http://www.retsch.com/) with a zirconia bead for 10 min at 20 Hz. Next, the samples were centrifuged at 15000 g for 10 min. The supernatant (500 ll) was diluted to 5.0 ml using water containing 0.1% acetic acid, and was applied to a 3 cc OASIS HLB cartridge (Waters, http://www.waters.com/) that had been equilibrated with 0.5% aqueous methanol containing 0.1% acetic acid. After pre-equilibrium with 5 ml of 0.5% aqueous methanol containing 0.1% acetic acid, metabolites were eluted with 3 ml of 90% aqueous methanol containing 0.1% acetic acid. The eluate was dried under vacuum and then suspended in 100 ll water containing the internal standard (0.5 mg/ml lidocaine). Insoluble residue was removed by filtration using an Ultrafree-MC filter with 0.2 lm pore size (Millipore, http://www.millipore.com/). The samples (3µl) were subsequently subjected to metabolome analysis by liquid chromatography coupled with electrospray quadrupole time-of-flight tandem mass spectrometry (LC-ESI-Q-TOF-MS) using an Acquity BEH ODS column (LC, Waters Acquity UPLC system; Q-TOF-MS, Waters Q-Tof Premier). C system; Q-TOF-MS, Waters Q-Tof Premier).
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