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).

RAW264.7细胞与载体（DMSO，'w/o'）或0.5、5.0 μM α-T-13′-羧酸共同孵育24小时。随后通过超高效液相色谱-串联质谱（UPLC-MS/MS）分析甘油三酯的脂肪酸分布。已上传UPLC-MS/MS结果的原始分析文件（.wiff和.wiff.scan格式）及样本列表的Excel文件。相关方法与结果发表于Liao等人的研究（Int J Mol Sci, 2023年5月25日；24(11):9229，doi:10.3390/ijms24119229）
从RAW264.7细胞沉淀中提取脂质，依次加入甲醇、磷酸缓冲盐溶液（PBS，pH 7.4）、氯仿和生理盐水（最终比例：34:14:35:17）。蒸发有机溶剂后，剩余脂质组分溶于甲醇，于-20℃保存，并通过超高效液相色谱-串联质谱（UPLC-MS/MS）分析。内标：1,2-二肉豆蔻酰-sn-甘油-3-磷脂酰胆碱（DMPC）、1,2-二肉豆蔻酰-sn-甘油-3-磷脂酰乙醇胺（DMPE）
甘油三酯（TGs）在Acquity UPLC BEH C8色谱柱（130 Å，1.7 μm，2.1×100 mm；Waters）上分离，所用系统为Acquity UPLC系统（Waters）（耦合至配备Turbo V离子源和电喷雾电离探针的QTRAP 5500质谱仪（Sciex））或ExionLC AD UHPLC系统（Sciex）（耦合至配备IonDrive Turbo V离子源和电喷雾电离探针的QTRAP 6500+质谱仪（Sciex））。简言之，两种液相色谱（LC）系统的流速均为0.75 mL/min，柱温为45℃。流动相由洗脱液A（乙腈/水，95/5，含2 mM醋酸铵）和洗脱液B（异丙醇）组成。分离TGs时，洗脱液A在6分钟内从90%降至70%，随后等度洗脱4分钟。[M+NH4]+加合离子碎裂为[M-脂肪酸阴离子]+离子的过程通过多反应监测（multiple reaction monitoring）进行检测。离子喷雾电压设置为5500 V，气帘气压力为30 psi（QTRAP 5500）或40 psi（QTRAP 6500+），碰撞气强度为低，加热毛细管温度为400℃。鞘气压力设置为60 psi，辅助气压力为70 psi。去簇电压设置为120 V，入口电压为10 V，碰撞能量为35 eV，碰撞池出口电压为26 V
仪器分别使用Analyst 1.6.2（QTRAP 5500，Sciex）或Analyst 1.7.1（QTRAP 6500+，Sciex）软件操作