| MS Description
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The collected sample tissues were weighed … The collected sample tissues were weighed and stored at −80°C until analysis. The frozen tissues of independent plants were homogenized in five volumes of 80% aqueous methanol containing 0.1% acetic acid, 0.5 mg/l of lidocaine, and d-camphor sulfonic acid (Tokyo Kasei, Tokyo, Japan) using a mixer mill (MM 300, Retsch) with a zirconia bead for 6 min at 20 Hz. Next, the samples were centrifuged at 15,000 g for 10 min and filtered (Ultrafree-MC filter, 0.2 μm; Millipore, Bedford, MA, USA). The sample extracts were then applied to an HLB μElution plate (Waters, Milford, MA, USA) that had been equilibrated with 80% aqueous methanol containing 0.1% acetic acid.
The eluates (3 μl) were subsequently subjected to metabolome analysis by LC coupled with electrospray quadrupole time-of-flight tandem MS using an Acquity BEH ODS column (LC-ESI-Q-Tof/MS, HPLC: Waters Acquity UPLC system; MS: Waters Q-Tof Premier).
The metabolome analysis and data processing were conducted according to a previously described method (Matsuda et al., 2009c, 2010a). Briefly, the metabolome data were obtained in the negative ion mode (m/z 100–2,000; dwell time: 0.45 s; interscan delay: 0.05 s, centroid), from which a data matrix was generated with the aid of MetAlign (De Vos et al., 2007; Lommen, 2009). In order to reduce a redundancy of the data matrix, fragment ions were removed by a following procedure. A metabolite signal was removed from the matrix when there is another intense peak eluted at similar retention
times [within the retention time threshold (<0.5 s)] with the highest correlation coefficient above the threshold value (>0.8). The analysis was conducted using five biological replicates of 20 accessions, from which a data matrix composed of 703 signals (peaks) was obtained (Table S1 in Supplementary Material). The number of signals would not reflect an exact number of detected metabolites due to the complex nature of the metabolome data.
To construct MS2T libraries, the extracts of five ecotypes were mixed and utilized for the MS2T data acquisition. The analyses were repeatedly conducted for four mixtures by previously described methods (Matsuda et al., 2009c). Each MS2T entry was assigned a unique accession code, such as ATH10n03690, in which ATH10n is the name of the library and 03690 is the entry number. All data obtained in this study are available at the PRIMe website1 (Akiyama et al., 2008). the PRIMe website1 (Akiyama et al., 2008).
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