2.7. LC-MS/MS Analysis

Tissue sample was grounded by liquid nitrogen. Then, tissue samples were firstly bath sonicated for 2 min with 400 μL ice-cold 75% to break up the cells. Next, 1 mL MTBE was added and the samples were shaken for 1 h at room temperature. Next, phase separation was induced by adding 250 μL water, letting it sit for 10 min at room temperature and centrifuging for 15 min at 14,000g, 4°C. Because of the low density and high hydrophobicity of MTBE, lipids and lipophilic metabolites are mainly extracted to the upper MTBE-rich phase. The lipid was transferred to fresh tubes and dried with air nitrogen.

Lipid analysis was performed on a Q Exactive orbitrap mass spectrometer (Thermo, CA). Mobile phase A is prepared by dissolving 0.77 g of ammonium acetate to 400 mL of HPLC-grade water, followed by adding 600 mL of HPLC-grade acetonitrile. Mobile phase B is prepared by mixing 100 mL of acetonitrile with 900 mL isopropanol.

Lipids were identified and quantified using LipidSearch 4.1.30 (Thermo, CA). Mass tolerance of 5 ppm and 10 ppm was applied for precursor and product ions. Retention time shift of 0.25 min was performed in “alignment.” M-score and chromatographic areas were used to reduce false positives.