Lipid extraction from biological samples

The lipids in lipid-raft and non-raft fractions were extracted using a modified MTBE extraction method. 100 μL of each lipid-raft was mixed with 100 μL H₂O and 480 μL of extraction solvent (MTBE: MeOH = 5:1, v/v, containing 1.5 μg of PC(15:0/18:1-d7), 0.5 μg of PE(15:0/18:1-d7), 0.25 μg of PG(15:0/18:1-d7), 0.5 μg of PS(15:0/18:1-d7), 0.5 μg of PI(15:0/18:1-d7), 0.05 μg of PA(15:0/18:1-d7), 0.2 μg of ceramide(d18:1-d7/15:0), 0.05 μg of SM(d18:1/18:1-d9), 0.02 μg of LPC(18:1-d7/0:0), 0.02 μg of LPE(18:1-d7/0:0), 10 μg of TG(15:0/18:1-d7/15:0)). 100 μL of each non-raft sample was mixed with 100 μL H₂O and 480 μL of extraction solvent (MTBE: MeOH = 5:1, v/v, containing 1.5 μg of PC(15:0/18:1-d7), 0.5 μg of PE(15:0/18:1-d7), 0.25 of μg PG(15:0/18:1-d7), 0.5 μg of PS(15:0/18:1-d7), 0.5 μg of PI(15:0/18:1-d7), 0.05 μg of PA(15:0/18:1-d7), 0.02 μg of ceramide(d18:1-d7/15:0), 0.01 μg of SM(d18:1/18:1-d9), 0.02 μg of LPC(18:1-d7/0:0), 0.02 μg of LPE(18:1-d7/0:0), 10 μg of TG(15:0/18:1-d7/15:0)). Each sample was vortexed for 30 s, followed by 10 min of sonication and 15 min of centrifugation at 13,000 × g. The upper organic layer was collected. Then, 200 μL of MTBE was added to the left aqueous layer for re-extraction. The re-extraction process was repeated twice, and the pooled organic layer was evaporated using a vacuum concentrator. The dried extract was reconstituted in 200 μL of DCM: MeOH (1:1, v/v) for lipid quantitative analysis.

For the quantification of cholesterol, 20 μL of each lipid-raft or non-raft sample was firstly diluted to 200 μL using H₂O. Then 100 μL of each sample was mixed with 100 μL H₂O and 480 μL extraction solvent (MTBE: MeOH = 5:1, v/v, containing 2 μg of cholesterol-d7). Each sample was vortexed for 30 s, followed by 10 min of sonication and 15 min of centrifugation at 13,000 × g. The upper organic layer was collected. Then, 200 μL of MTBE was added to the left aqueous layer for re-extraction. The re-extraction process was repeated twice, and the pooled organic layer was evaporated using a vacuum concentrator. The dried extract was reconstituted in 100 μL of DCM: MeOH (1:1, v/v) for LC-MS analysis. An external calibration curve of cholesterol was also measured with a linear range from 0.5 μg mL⁻¹ to 200 μg mL⁻¹. The internal standard cholesterol-d7 (20 μg mL⁻¹) was added into each calibration sample. Finally, the absolute concentrations of cholesterol in lipid-raft or non-raft samples were quantified according to the measured peak area of cholesterol in comparison to the internal standard (cholesterol-d7) by interpolation from the calibration curve.

For the quantification of cholesterol in HEK-293T cells, 20 μL of each cell sample was firstly diluted to 200 μL by using H₂O. Then, 100 μL of each sample was mixed with 100 μL of H₂O and 480 μL of extraction solvent (MTBE: MeOH = 5:1, v/v, containing 1 μg of cholesterol-d7). The rest procedures were the same as the preparation of lipid-raft samples. Finally, the dried extract was reconstituted in 100 μL of DCM: MeOH (1:1, v/v). A calibration curve of cholesterol was also measured with a linear range of cholesterol from 1 μg mL⁻¹ to 100 μg mL⁻¹, and cholesterol-d7 (10 μg mL⁻¹) was also added into each calibration sample as internal standard. The total protein concentration of each cell sample was measured at beginning by a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225). The cellular cholesterol concentration was normalized to the corresponding protein concentration of each sample.