C18 plasm 20 4 pc

This step was performed as described previously (40 (link)), modified and extended to also allow quantification and fragmentation of glycerophosphocholines and glycerophosphoethanolamines. Internal standards were added to cell homogenates, and lipids were extracted via the Folch procedure (41 (link)). Dried lipid extracts were dissolved in HPLC starting condition, separated by reversed-phase HPLC on a Dionex Ultimate 3000 HPLC (Thermo Fisher Scientific), and quantified with a Velos Pro Dual-Pressure Linear Ion Trap Mass Spectrometer (Thermo Fisher Scientific). Baseline corrected data were integrated in MZmine 2 (42 (link)), quantified, and visualized as described previously (40 (link)) using custom-made scripts in R (https://www.R-project.org/). Molecular PE, PC, plasmanyl-PE and plasmenyl-PE species were identified by their retention time, monoisotopic mass-to-charge ratio, isotope pattern, and fragmentation behavior (SI Appendix, Fig. S5 and Materials and Methods), which was cross-validated with single lipid standards commercially available from Avanti Polar Lipids: C16-18:1 PC, C18(Plasm)-22:6 PC, C16-18:1 PE, C18(Plasm)-18:1 PC, C18(Plasm)-18:1 PE, and C18(Plasm)-20:4 PC.

Polar lipid classes composition was first analyzed via thin-layer chromatography (TLC) using silica gel plates (Sorbfil, Krasnodar, Russia) and a solvent system of dichloromethane/diethyl ether/NH₃ (65:35:4, v/v/v). Then, the TLC plate was sprayed with 10% H₂SO₄ in MeOH and heated at 240 °C for 10 min after air-drying. The PL classes were detected by comparing their Rf values with each standard sample. Polar lipid standards PC (16:0–20:4), PE (16:0–20:4), PS (16:0–20:4), PI (18:0–20:4), C18(Plasm)-20:4 PC, and C18(Plasm)-20:4 PE were purchased from Avanti Polar Lipids Co. (Alabaster, AL, USA).

All solvents were of HPLC or liquid chromatography–mass spectrometry (LC–MS) grade. Polar lipid standards (16:0–20:4 PC, 16:0–20:4 PE, 16:0–20:4 PS, 18:0–20:4 PI, C18(Plasm)-20:4 PC, C18(Plasm)-20:4 PE, C16-18:1 PC) were purchased from Avanti Polar Lipids Co. (Alabaster, USA). Neutral lipid standards (stearyl oleate, cholesterol oleate, 1-O-hexadecyl-2,3-dihexadecanoyl-rac-glycerol, glycerol trioleate, oleic acid, cholesterol) and column silica gel (high-purity grade, 70–230 mesh) were from Sigma-Aldrich Co. (St. Louis, USA). A mixture of PUFA methyl esters No. 3 from menhaden oil was obtained from Supelco (Bellefonte, USA). 2-Amino-2-methyl-1-propanol and trifluoroacetic anhydride (Fluka, Germany) were used for the synthesis of 4,4-dimethyloxazoline (DMOX) derivatives of FAs. The precoated silica gel thin-layer chromatography (TLC) plates with a silica sol binder on aluminum foil (PTLC-AF-V) were provided by Sorbfil (Krasnodar, Russian Federation).