Analysis of the phosphatidylserine fatty acid composition in α-T-13′-COOH-treated macrophages

RAW264.7 cells were incubated with vehicle (DMSO, 'w/o') or 0.5 or 5.0 µM α-T-13′-COOH for 24 h. The fatty acid distribution of phosphatidylserine was then analyzed by UPLC-MS/MS.
Raw analyst files (.wiff and .wiff.scan) of the UPLC-MS/MS results were uploaded, together with an Excel file for the sample list.
The methods and results were published in Liao et al., Int J Mol Sci, 2023 May 25;24(11):9229. doi: 10.3390/ijms24119229.
Lipids were extracted from RAW264.7 cell pellets by the successive addition of methanol, PBS (pH 7.4), chloroform, and saline (final ratio: 34:14:35:17). After the evaporation of the organic solvent, the remaining lipid fraction was dissolved in methanol, stored at −20 °C, and analyzed by UPLC-MS/MS. Internal standards: 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphatidyl-ethanolamine (DMPE).
Phospholipids (PC, PE, PI, PS, PG) were separated on an Acquity UPLC BEH C8 column (130 Å, 1.7 μm, 2.1 × 100 mm; Waters, Milford, MA, USA) using an Acquity UPLC system (Waters), which was coupled to a QTRAP 5500 mass spectrometer (Sciex, Framingham, MA, USA) equipped with a Turbo V Ion Source and an electrospray ionization probe. Chromatographic separation was performed at a flow rate of 0.75 mL/min and at a column temperature of 45 °C. The mobile phase was composed of eluent A (acetonitrile/water, 95/5, with 2 mM ammonium acetate) and eluent B (water/acetonitrile, 90/10, with 2 mM ammonium acetate). The gradient was ramped from 70% to 80% A within 5 min and to 100% A within 2 min, followed by isocratic elution for another 2 min. Eluted phospholipids were detected upon the fragmentation of parental ions (PC: [M+OAc]−, all other phospholipids: [M-H]−) to fatty acid anions derived from sn-1 and sn-2 positions by multiple reaction monitoring using a QTRAP 5500 mass spectrometer. The ion spray voltage was set to −4500 V, the curtain gas to 30 psi, the collision gas to medium, and the heated capillary temperature to either 350 °C (PC), 500 °C (PI), 550 °C (PS, PG), or 650 °C (PE). The sheath gas pressure was set to 45 (PS) or 55 psi (PC, PE, PI, PG) and the auxiliary gas pressure was set to either to 75 psi (PC, PE, PI, PG) or 80 psi (PS). The declustering potential was set to −40 V (PS), −44 V (PC), −45 V (PG), or −50 V (PE, PI), the entrance potential to −10 eV (PC; PE, PI, PS, PG), the collision energy to −38 eV (PE), −46 eV (PC), −52 eV (PG), −56 eV (PS), or −62 eV (PI), and the collision cell exit potential to −11 V (PC, PI), −12 V (PE), −18 V (PG), or −20 V (PS).
The instruments were either operated with Analyst 1.6.2 (QTRAP 5500, Sciex) or Analyst 1.7.1 (QTRAP 6500+, Sciex).