Lipidomics by LC-MS/MS

After incubation, lipids were extracted as described previously [30]. Lipid extracts were dried under nitrogen and dissolved in 100 μL chloroform/methanol (1:1) and injected (10 μL) on a hydrophilic interaction liquid chromatography (HILIC) column (2.6 μm HILIC 100 Å, 50 × 4.6 mm, Phenomenex, Torrance, CA, USA). Lipid classes were separated by gradient elution on an Infinity II 1290 UPLC (Agilent, Santa Clara, CA, USA). At a constant flow rate of 1 mL/min, acetonitrile/acetone (9:1, v/v) was used as solvent A. Solvent B consisted of a mixture of acetonitrile/H₂O (7:3, v/v) with 10 mM ammonium formate. Both solvents contained 0.1% formic acid. The gradient was as follows (time in min, %B): (0, 0), (1, 50), (3, 50), (3.1, 100), (4, 100). Samples were injected without re-equilibration of the column. The column effluent was connected to a heated electrospray ionization source of an Orbitrap Fusion mass spectrometer (Thermo Scientific) operated at –3,600V in the negative ionization mode. Temperatures for the vaporizer and ion transfer tube were 275°C and 380°C, respectively. Full scan MS1 measurements in the mass range from 450 to 1,150 u were collected in the Orbitrap at a resolution of 120,000. Parallelized data-dependent MS2 experiments were done with higher-energy collisional dissociation fragmentation set at 30V, using the dual-stage linear ion trap to generate up to 30 spectra per second.