| MS Description
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The frozen tissues were homogenized in fiv … The frozen tissues 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) using a mixer mill (MM 300, Retsch) with a zirconia bead for 6 min at 20 Hz. Following centrifugation at 15,000g for 10 min and filtration (Ultrafree-MC filter, 0.2 μm, Millipore), the sample extracts were applied to an HLB μElution plate (Waters) equilibrated with 80% aqueous methanol containing 0.1% acetic acid. The eluates (3 μL) were subjected to metabolome analysis using LC-ESI-Q-TOF/MS.
Metabolome analysis was performed with an LC-ESI-Q-TOF/MS system equipped with an ESI interface (HPLC: Waters Acquity UPLC system; MS: Waters Q-TOF Premier) operated under previously described conditions (Matsuda et al., 2009). In the negative ion mode, the MS conditions were as follows: capillary voltage: +3.0 keV; cone voltage: 22.5 V; source temperature: 120°C; desolvation temperature: 450°C; cone gas flow: 50 L/h; desolvation gas flow: 800 L/h; collision energy: 2 V; detection mode: scan (m/z 100–2,000; dwell time: 0.45 s; interscan delay: 0.05 s, centroid); dynamic range enhancement mode: on. The scans were repeated for 19.5 min in a single run. ere repeated for 19.5 min in a single run.
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