Lipid extraction and LC–MS/MS lipidomics analysis

Lipids extraction and analysis was performed as previously outlined⁴⁹. Briefly, frozen cell pellet lipids were extracted using the Folch method with Chloroform/Methanol/Water (2:1:1 ratio). Lipids were dried using a SpeedVac (Savant SP131DDA, Thermo Scientific, Runcorn, UK) and re-suspended in Chloroform/Methanol (1:1), prior to injection into a Shimadzu Prominence 20-AD system (Shimadzu, Kyoto, Japan). Chromatographic separation was achieved using a Waters Acquity UPLC C4 (100 × 1 mm, 1.7 μm particle size) column (Milford, MA, U.S.A.). The column was kept a 45 °C and 7 μl of samples were eluted using a mobile phase composed of solvent A (water) and B (acetonitrile), each containing 0.025% formic acid. The gradient started at 45% B for 5 min, then increased to 90% B for 5 min, and 100% B was reached after an additional 10 min and held for 7 min before re-equilibration at 45% B for 5 min. The flow rate was maintained at 100 μl/min. Accurate mass (with an error below 5 ppm) was acquired on an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Source parameters for positive polarity were: capillary temperature 275 °C; source heater temperature 200 °C; sheath gas 10 AU; aux gas 5 AU; sweep gas 5 AU. Source voltage was 3.8 kV. Full scan spectra in the range of m/z 340–1500 were acquired at a target resolution of 240,000 (FWHM at m/z 400). Manual inspection of the results was carried out using Xcalibur and further processed using Lipid Data Analyzer (LDA) 2.7.0_2019 software⁵⁰.

Targeted analysis of lysolipids, Cer, and dhCer, was performed using a QTRAP 6500 LC–MS/MS System (AB SCIEX) operating in MRM mode. Quantification of multiple species of ceramides was carried out by the integration of the peak area as normalized against the peak area of the non-endogenous odd-chain ceramide C17:0. C17 was present at a known concentration and served as the internal standard (IS). Collision energy (CE) was optimized previously.

Thirtyfive lipid subclasses were identified, including alkenyl-acylphosphatidylcholine (P-PC), alkenyl-acylphosphatidylethanolamine (P-PE), alkyl-acylglycerol (O-DG), alkyl-acylphosphatidylcholine (O-PC), alkyl-acylphosphatidylethanolamine (O-PE), alkyl-triacylglycerol (O-TG), cardiolipin (CL), ceramides (Cer), cholesterol (CH), cholesterol ester (CE), diacylglycerol (DG), dihydroceramides (dhCer), dihydrosphingomyelin (dhSM), free fatty acids (FA), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), sphingosine (SG), triacylglycerol (TG), lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), lyso phosphatidylinositol (LPI), phosphatidylserine (LPS), alkenyl-lysophosphatidylcholine (P-LPC), alkenyl-lysophosphatidic acid (P-LPA), alkyl-lysophosphatidylethanolamine (O-LPE), alkyl-lysolysophosphatidylcholine (O-LPC), phosphatidylinositolmonophosphate (PIP), phosphatidylinositoldiphosphate (PIP2), phosphatidylinositoltriphosphate (PIP3).

Lipid relative quantitation levels were calculated using the R-studio (v3.2.4) software⁵¹ (https://www.R-project.org) with in-house built scripts. Statistical comparison between the P cells and PCSCs was performed using the paired t-test (p < 0.005), principal component analysis (PCA), and log₂ ratio transformation of PCSCs versus parental lipid levels.