4.4. Lipidomic Analysis

Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for targeted lipidomic analysis, similar to our earlier studies [41,42]. Briefly, an Agilent 1200 series HPLC system with a Poroshell 120 EC-C18 column (2.1 × 100 mm 2.7 micron, Agilent Technologies, Mulgrave, VIC, Australia; heated to 50 °C) was coupled to an electrospray ionization Qtrap 4000 mass spectrometer (ABS SCIEX, Framingham, MA, USA). A binary solvent system with solvent A (60% H20, 20% MeOH and 20% tetrahydrofuran (THF) (v/v/v) with 10 mM ammonium formate) and solvent B (75% THF, 20% MeOH and 5% H₂O (v/v/v) with 10 mM ammonium formate) was used. A total of 4 lipidomic acquisition methods were used in this study. Two of which were for general lipid profiling, which also monitored lipid classes other than PC and PE and are referred to as general method 1 and 2 (Supplementary Table S7). Two additional methods were developed specifically for flux analysis and focused on PC, PE, LPC, LPE, and sphingomyelin. One flux analysis method included +0 and +4 m/z for each lipid species detected (+4 method) while the other had +0, +3, +6 and +9 m/z (+369 method, Supplementary Table S8). The former was used for D4-ethanolamine tracer experiments and the latter for D9-choline and D3-methionine tracer experiments. The run time (20 min) and the detection modes for precursor/product ion pair for each lipid class were identical for all four acquisition methods. These methods had slightly different mass spectrometric voltage settings, as recorded in Supplementary Tables S7 and S8. For general method 1 and the flux analysis methods, the solvent gradient was set to: 90% A, 10% B at time 0; 10 to 100% B from 0 to 13 min; 100% until 16 min; 100 to 10% from 16 to 17 min and 10% B for another 3 min. For general method 2, the solvent gradient was: 90% A, 10% B at time 0; 10 to 20% B from 0 to 0.1 min; 20 to 35% B from 0.1 to 3 min; 35 to 50% B from 3 to 3.5 min; 50 to 62% B from 3.5 to 11 min; 62 to 89% B from 11 to 11.1 min; 89 to 92% B from 11.1 to 14.9 min; 92 to 100% B from 11.9 to 15 min, then 100% until 16.5 min; 100 to 10% from 16.5 to 17 min and 10% B for another 3 min. We adopted this stepped solvent gradient because it allowed a better resolution for lipid peaks of interest. Plasmenyl ether phospholipid species were identified by acid hydrolysis of the vinyl alcohol bond and subsequent removal of the alkenyl chain, as described in our earlier work [43]. AB SCIEX Multiquant software version 2.1.1 (Framingham, MA, USA) was used for peak quantifications and integrations as well as the calculation of lipid concentrations.