Lipid Extraction and LC-MS/MS Analysis

The endogenous bound lipids from SPNS2 proteins were extracted by adding chloroform-methanol (2:1, v/v) as previously described.⁴⁸ (link) The extracted lipids were dried and resuspended in 80% methanol. For LC-MS/MS analysis, the lipids were separated on a C18 column (Acclaim PepMap 100, C18, 75 μm × 15 cm, Thermo Fisher Scientific) using a Dionex UltiMate 3000 RSLC nano LC System using a previously described mobile phase and gradient.⁴⁹ (link) The column eluent was loaded to Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). The Orbitrap Eclipse was operated in negative mode using data-dependent acquisition. The detailed mass spectrometer parameters are as follows: spray voltage, -1.9 kV; capillary temperature, 320°C; mass range, 250-1500; full MS resolution, 120K; MS/MS resolution, 15K; NCE, 25/30/35; cycle time, 1.5s. The data was analysed by LipidSearch 4.2 software (Thermo Fisher Scientific).