Lipid Profiling by HILIC-LC-MS

Total lipid extracts were analyzed by hydrophilic interaction liquid chromatography using a high-performance liquid chromatography (HPLC) Ultimate 3000 Dionex (Thermo Fisher Scientific, Bremen, Germany) with an autosampler coupled online to a Q-Exactive hybrid quadrupole mass spectrometer (Thermo Fisher, Scientific, Bremen, Germany). The solvent system and instrumental settings were set as described previously [28 (link)]. To perform the HILIC-LC-MS analyses, 10 µg of total lipid extract, 2 µL of phospholipid standards mix (0.01 µg dimyristoylphosphatidylcholine (dMPC), 0.01 µg dimyristoylphosphatidylethanolamine (dMPE), lysophosphatidylcholine (LPC) 0.01 µg, 0.04 µg dipalmitoylphosphatidylinositol (dPPI), 0.006 µg dimyristoylphosphatidylglycerol (dMPG), 0.02 µg dimyristoylphosphatidylserine (dMPS), 0.04 µg tetramyristoylcardiolipin (tMCL), 0.01 µg sphingomyelin (SM(17:0/d18:1)), 0.04 µg dimyristoylphosphatidic acid (dMPA)) and 88 µL of eluent (40% of mobile phase A and 60% of mobile phase B) were mixed and injected into the Ascentis Si column HPLC Pore column (15 cm × 1 mm, 3 µm, Sigma-Aldrich, St Louis, USA), with a flow rate of 40 µL minutes⁻¹ at 30 °C. Acquisition in the Orbitrap® mass spectrometer was performed in both positive (electrospray voltage 3.0 kV, Thermo Scientific, Waltham, USA) and negative (electrospray voltage −2.7 kV) modes. For lipidomic analysis, phospholipid peak integration and assignments were performed using MZmine version 2.32 (Boston, USA) [29 (link)]. For all assignments, ions within 5 ppm of the lipid exact mass were considered. Analysis of the MS/MS spectra acquired in the positive ion mode was performed to confirm the identity of the molecular species belonging to the MGMG, DGMG, MGDG, DGDG, DGTS, PC and LPC classes. The MS/MS spectra acquired in the negative ion mode were used to confirm the identity of SQDG, SQMG, LPE, PE, LPG, PG, LPI, PI, lysophosphatidic acids (LPA) and phosphatidic acids (PA). Negative ion mode MS/MS data were used to identify the fatty acid carboxylate anion fragments RCOO⁻, which allowed the assignment of the fatty acyl chains esterified to the PL precursor. All the ions detected and MS/MS fragmentation patterns characteristic of the lipid classes detected and analyzed in the present study, acquired both in positive and negative ion modes, are available online as Supplementary Information (Supplementary Table S1 and Supplementary Figure S1). Normalization of the data was performed by dividing the peak areas of the extracted ion chromatograms (XICs) of the polar lipid precursors of each class (listed in Supplementary Table S1) by the peak area of the internal standard selected for the class.