| MS Description
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Two sample preparation methods were employ … Two sample preparation methods were employed for comparison. For the single-grain-based analysis, a visually normal seed was selected from each of the 12 individual plants under a microscope. After the estimation of seed volume as described above, each seed was placed in a separate well of a 96-well plate using a Vacuseed (Polaris Instruments Ltd, Cambridge, U.K.) with a 3-mm zirconia bead. The metabolites were extracted using 500 µL extraction solvent (80% methanol, 0.1% formic acid, 16.8 nmol/L lidocain, and 105 nmol/L 10-camphorsulfonic acid as internal standards) with a Multi-beads Shocker (Shake Master NEO, Bio Medical Science) at 1000 rpm for 10 min. After centrifugation, 250 µL of the extract was transferred to a new plate, dried, dissolved in 250 µL of ultra-pure water, and filtered using Whatman® UNIFILTER® plates 384 (GE Healthcare). The sample preparation process was automatically performed by a liquid handling system (Microlab Star Plus, Hamilton) [see widely targeted metabolomics section in PRIMe (http://prime.psc.riken.jp/) (Sakurai et al. 2013)]. 1 µL of the solution extract (final concentration = ca. 40 µg/mL) prepared from a seed (ca. 20 µg in the case of diploid) was subjected to widely targeted metabolomics using LC-QqQ-MS (UPLC-TQS, Waters).<br />
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For the weight-based analysis, approximately 200 seeds, weighing 4 ± 0.4 mg, from each of 12 plants, were collected using a Seed Spoon (BMS-SS200, Bio Medical Science). Seeds were accurately weighed, placed in a 2-mL tube with a 5-mm zirconia bead and proportional volume of the extraction solvent (4.0 mg/mL), and crushed using a Multi-beads Shocker at 1000 rpm for 10 min. After centrifugation, the extracts were diluted to 40 µg/mL with the extraction solvent. A 250 µL volume of the extract was transferred to a 96-well plate, dried, dissolved in 250 µL of ultra-pure water, and filtered using Whatman® UNIFILTER® plates 384. 1 µL of solution extract (40 µg/mL) was injected into an LC-QqQ-MS (UPLC-TQS, Waters) (Fig. 2). an LC-QqQ-MS (UPLC-TQS, Waters) (Fig. 2).
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