Sample preparation and lipidomic analysis with LC-MS

Cells were seeded in 6 well-plates and incubated for 24h at 37°C and 5% CO₂. Cells were then incubated in the presence of either PunA 2.5-5-10 μM, α-linolenic acid 10 μM, ferrostatin-1 (fer1) 10 μM or a combination of PunA 10 μM and fer1 10 μM. After 4h, cells were washed twice with cold PBS and lysed in 1 mL of 0.1% SDS lysis buffer. Protein concentration was measured with the Pierce bicinchoninic acid protein assay. Cell lysates were subjected to lipid extraction according to the method of Bligh and Dyer.⁷⁸ An internal standard composed of not naturally occurring lipid species was added as follows: cholesteryl ester (CE) 17:0, CE 22:0, cardiolipin (CL) 14:0/14:0/14:0/14:0, triglyceride (TG) 51:0, TG 57:0, diacylglycerol (DG) 28:0, DG 40:0, ceramide (Cer) d18:1;O2/14:0, Cer d18:1;O2[D7]/18:0, hexosylceramide (HexCer) 18:1;O2/12:0, HexCer 18:1;O2[D5]/18:0, free cholesterol FC[D7], phosphatidylcholine (PC) 14:0/14:0, PC 22:0/22:0, phosphatidyl-ethanolamine (PE) 14:0/14:0, PE 20:0/20:0 (di-phytanoyl), phosphatidylserine (PS) 14:0/14:0, PS 20:0/20:0, phosphatidylinositol (PI) 18:1[D7]/15:0, sphingomyelin (SM) 18:1;O2/12:0, SM 18:1;O2/18:1[D9], lysophosphatidylcholine (LPC) 13:0, LPC 19:0 and lysophosphatidylethanolamine (LPE) 13:0. The chloroform phase was recovered by a pipetting robot (Tecan Genesis RSP 150) and vacuum dried. The residues were dissolved in chloroform/methanol/2-propanol (1:2:4 v/v/v) with 7.5 mM ammonium formate or with 0.005% methylamine (for analysis of CL, HexCer, and PS). Lipids were analyzed using direct flow injection analysis (FIA) on a Fourier-Transform mass spectrometer (FIA-FTMS with high mass resolution).^80,81 The mass spectrometer was equipped with a heated electrospray ionization source and a standard-sized hybrid quadrupole-Orbitrap (Thermo Fisher Scientific). The ion source was operated using a spray voltage of 3.5 kV, S-lens RF level 50, a capillary temperature of 250°C, an aux gas heater temperature of 100°C, and settings of 15 for sheath gas and 5 for aux gas. 50 μl of reconstituted sample extracts were injected by a PAL autosampler (CTC Analytics, Zwingen) equipped with an UltiMate 3000 isocratic pump (Thermo Fisher Scientific). Chloroform/methanol/2-propanol (1:2:4 v/v/v) was delivered at an initial flow rate of 100 μl/min until 0.25 min followed by 10 μl/min for 2.5 min and a wash out with 300 μl/min for 0.5 min. Enhanced Fourier-transform was applied for signal processing. All data were acquired in profile mode. TG, DG and CE were recorded in positive ion mode FTMS in m/z range 500–1000 for 1 min with a maximum injection time (IT) of 200 ms, an automated gain control (AGC) of 1∗10⁶, 3 microscans, and a target resolution of 140,000 (at m/z 200). Negative ion mode FTMS data were recorded in m/z range 400–650 for LPE and LPC and m/z range 520–960 for PC, PE, PI, PS Cer, HexCer and SM quantification. Cardiolipins were analyzed as [M-H]^- in negative ion mode m/z range 1100–1600. Multiplexed acquisition was used for the [M+NH₄]⁺ of free cholesterol (FC) and FC[D7] for 0.5 min acquisition time, with a normalized collision energy of 10%, an IT of 100 ms, AGC of 1∗10⁵, isolation window of 1 m/z, and a target resolution of 140,000. All FTMS data were processed using the ALEX software,⁸² which includes peak assignment and intensity picking. Extracted data were exported to Microsoft Excel and processed by generic macros. The quantification was performed by multiplication of the spiked IS amount with analyte-to-IS ratio. Lipid species were annotated according to the proposal for shorthand notation of lipid structures derived from MS.⁸³

All analyses were performed in R (csv4.2.2).⁸⁴ Lipid species with a frequency of 70% missing values were filtered out and lipid species abundances were log2 transformed. To explore the lipidomic data, Principal Component Analysis (PCA) was conducted on all log-transformed lipid species abundances for all cell lines by using the MissMDA R package (version 1.18),⁸⁵ to impute the remaining missing values by regularized iterative PCA algorithm. In order to identify the lipid species whose abundance changes with treatments, Differential Expression Analysis (DEA) was conducted on the abundances of all lipid species for all cell lines by performing a regression with the treatment effect based on the proDA package (version 1.10.0).⁸⁶ This method accounts for missing values without imputation by implementing a probabilistic dropout model. Significance was then established with a Wald test on the model coefficient of interest and p-values were adjusted with False Discovery Rate (FDR) for multiple testing.