Lipid Analysis

Cells were harvested by scraping from the insert membrane in 100 μl PBS + cOmplete Mini protease inhibitor and PhosSTOP phosphatase inhibitor (Sigma, at the concentrations indicated by the manufacturer), and the lipid fraction was extracted using 2 vol (chloroform): 1 vol (methanol): 1 mM Butylhydroxyanisol (BHA). Tubes were mixed vigorously and centrifuged at 4°C for 5 min at 3,000 rpm. The organic phase was recovered and evaporated using a Speedvac and stored in liquid nitrogen. The extracted lipids were separated as previously described (Guilbault et al., 2008, 2009; Garic et al., 2017, 2020). Malondialdehyde measurement (MDA), was performed by the TBARS assay as described previously (Guilbault et al., 2009; Garic et al., 2017, 2020). Nitrotyrosine and total ceramides were quantified as previously described in detail (Guilbault et al., 2008, 2009; Garic et al., 2017, 2020). Total ceramides were measured by ELISA after TLC purification, whereas quantification of the specific ceramides species were done using mass spectroscopy using the total ceramide pool purified on TLC. Mouse lung lipids was collected and analyzed by mass spectrometry after extraction as described in (Veltman et al., 2016).

LC-MS/MS was carried out using a QTrap 5500 mass spectrometer (AB sciex) coupled to a Dionex UltiMate 3000 LC-system. The separation column was Kinetex 2.1 × 50 mm C18, guarded with a SecurityGuard 4 × 2.0 mm C18 guard pre-column (Phenomenex). The mobile phases were MilliQ water (A) and methanol (B), both with 0.01% acetic acid, with the following gradient: first minute 20% B, increase to 35% B in 3 min and further increase to 99% B in 15 min and 100% B in 17.1. The 100% B was held for 0.9 min, then the gradient was decreased back to 20% B in 1 min and column was equilibrated at 20% B for another minute. The total run time was 20 min. MS was operated under the following conditions: the collision gas flow was set to medium, the drying temperature was 400°C, the needle voltage was −4,500 V, the curtain gas was 30 psi, ion source gas 1 was 40 psi, and the ion source gas 2 was 30 psi. Lipid standards were purchased from Sigma Aldrich and Avanti Polar Lipids.