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20 mg frozen tissue was weighed and placed in extraction solution (600 µl methanol:chloroform at 66:33 (v/v)) and homogenized at 2,823g for 20 s twice. Homogenized samples were further sonicated for 15 min and 200 μl chloroform and water were separately added. After 1 min of vortexing, extracts were stored at 4 °C for 10 min, followed by centrifugation at 4 °C, 18,213g for 20 min. Upper aqueous supernatant and lower lipophilic solution were separately transferred to clean microcentrifuge tubes and dried using a speed vac evaporator and nitrogen evaporator, respectively. Powdered samples were stored at −80 °C till LC–MS analysis. On the same day as LC–MS analysis, dried polar metabolites were immediately re-dissolved in water:acetonitrile at 25:75 solution for polar metabolite metabolomics. Similarly, dried lipid spices were re-dissolved in isopropanol:acetonitrile at 10:90 (v/v) for lipidomics.

LC separations were carried out on an Acquity UPLC BEH C18 column (Waters) by UPLS system (Waters). Column temperature and flow rate were set to 35 °C and 0.35 ml min−1, respectively. The mobile phase A was 10 mM ammonium acetate in acetonitrile:water at 40:60 (v/v) and the mobile phase B was 10 mM ammonium acetate in acetonitrile:isopropanol at 10:90 (v/v). The gradient elution started at 60% A:40% B and B was increased linearly to 65% at 5 min, 70% at 12 min and 99% at 15 min. Then, 1% A:99% B was kept for 2 min and column was equilibrated for 3 min. Pooled QC samples were also measured to check data. For each sample, 5 μl were injected with a partial loop mode for positive and negative ionization polarity modes separately from auto-samplers at 4 °C.

MS analysis was performed on a triple TOF 5600 MS/MS System (AB Sciex). Lipid species were analyzed in the range m/z 50–1,500. The following specific settings were used: ion spray voltage, 5,500 V; source temperature, 500 °C; nebulizer gas pressure, 60 psi; drying gas pressure, 60 psi; and curtain gas pressure, 30 psi.