Analysis of the triglyceride 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 triglyceride 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).
TGs were separated on an Acquity UPLC BEH C8 column (130 Å, 1.7 μm, 2.1 × 100 mm; Waters) using either an Acquity UPLC system (Waters), which was coupled to a QTRAP 5500 mass spectrometer (Sciex) equipped with a Turbo V Ion Source and an electrospray ionization probe, or an ExionLC AD UHPLC system (Sciex), which was coupled to a QTRAP 6500+ mass spectrometer (Sciex) equipped with an IonDrive Turbo V Ion Source and an electrospray ionization probe. In brief, both LC systems were operated at a flow rate of 0.75 mL/min and a column temperature of 45 °C. The mobile phase consisted of eluent A (acetonitrile/water, 95/5, with 2 mM ammonium acetate) and eluent B (isopropanol). For the separation of TGs, eluent A was reduced from 90% to 70% within 6 min followed by isocratic elution for 4 min. The fragmentation of [M + NH4]+ adduct ions to [M-fatty acid anion]+ ions was measured by multiple reaction monitoring. The ion spray voltage was set to 5500 V, the curtain gas to 30 psi (QTRAP 5500) or 40 psi (QTRAP 6500+), the collision gas to low, and the heated capillary temperature to 400 °C. The sheath gas pressure was set to 60 psi and the auxiliary gas pressure was set to 70 psi. The declustering potential was set to 120 V, the entrance potential to 10 V, the collision energy to 35 eV, and the collision cell exit potential to 26 V.
The instruments were either operated with Analyst 1.6.2 (QTRAP 5500, Sciex) or Analyst 1.7.1 (QTRAP 6500+, Sciex).