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To extract intracellular lipids, 2 x 106 cells were quickly twice washed with PBS buffer and quenched in liquid nitrogen for 1 min. After the cells were scraped into 200 μL of cold methanol-water mixture (8:2, v/v), the extract was transferred into a 1.5 mL Eppendorf tube. This extraction step was repeated twice. The extract was vigorously mixed with 200 μL of water and 400 μL of chloroform for 1 min and then, kept at 4 °C for 30 min. After centrifuge at 21,191×g and 4 °C for 20 min, 350 μL of lower lipophilic solution was transferred into a new tube and dried under nitrogen gas. Powdered samples were stored at −80 °C until LC-MS analysis. Dried lipid metabolites were re-dissolved in 200 μL of isopropanol containing SPLASH LIPIDOMIX internal standard mix (Avanti Polar Lipids) and palmitic acid (U-13C16, 98%, Cambridge Isotope Laboratories, Inc.). After dilution 5-fold for positive mode and 2-fold for negative mode with isopropanol-water mixture (8:2, v/v), 2 μL of each sample was injected into UPLC-QTOF-MS system.

Intracellular lipids were separated using an Acquity UPLC CSH C18 column (100 mm × 2.1 mm, 1.7 μm particle size; Waters) at 55 °C and a flow rate of 0.4 ml/min. The mobile phase comprised 10 mM ammonium formate in water-acetonitriler mixture (4:6, v/v) containing 0.1% formic acid (solvent A), and isopropanol-acetonitrile mixture (9:1, v/v) containing 0.1% formic acid (solvent B) for positive ion mode. For negative ion mode, solvent A consisted of 10 mM ammonium acetate in water-acetonitriler mixture (6:4, v/v) and solvent B consisted of isopropanol-acetonitrile mixture (9:1, v/v). The linear gradient program was as follows: 40–43% B from 0 min to 2 min, 43–50% B from 2 min to 2.1 min, 50–54% B from 2.1 min to 12 min, 54–70% B from 12 min to 12.1 min, 70–99% B from 12.1 min to 18 min, 99–40% B from 18 min to 18.1 min, and 40% B for 2 min to equilibrate for the next run.

MS spectral data acquisition was performed in positive and negative electrospray ionization (ESI) modes with an m/z range of 50–1,200. The data acquisition parameters as follows: capillary voltages of +2,000 and −1,000 V for positive and negative ion modes, a sample cone of 30 V, source offset of 80 V, source temperature of 120 °C, desolvation temperature of 600 °C, cone gas flow of 150 and 50 L/hour for positive and negative ion mode, scan time of 0.1 s. To maintain mass accuracy, leucine-enkephalin was used as the lock mass generating an [M+H]+ ion at m/z 556.2771 in positive mode, and an [M-H]- ion at m/z 554.2771 in negative mode. MS fragmented spectral data were acquired using product ion scan mode for lipid classes including acylcarnitine (ACar), free fatty acid (FFA), lysophosphatidylethanolamine (LysoPE), lysophosphocholine (LysoPC), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin (CL), ceramide (Cer), sphingomyelin (SM), glucosylceramide (GlcCer), diacylglycerols (DAG) and triacylglycerols (TAG).