Lipidomic standards were from Avanti Polar Lipids (Alabaster, AL, USA). Solvents for extraction and MS analyses were Optima LC-MS grade (Thermo Fisher Scientific, Waltham, MA, USA) and liquid chromatographic grade (Merck, Darmstadt, Germany). All other chemicals were the best available grade purchased from Sigma (Steinheim, Germany) or Merck (Darmstadt, Germany).
MS analyses were performed on an Orbitrap Fusion Lumos instrument (Thermo Fisher Scientific, Bremen, Germany) equipped with a robotic nanoflow ion source (TriVersa NanoMate, Advion BioSciences, Ithaca, NY, USA) using chips with a spraying nozzle diameter of 5.5 µm. The back pressure was set at 1 psi. The ionization voltages were +1.3 kV and −1.9 kV in positive and negative modes, respectively, whereas it was +1.5 kV in acquisitions with polarity switching. The temperature of the ion transfer capillary was 260 °C. Acquisitions were performed at mass resolution Rm/z 200 = 240,000 in full scan mode. In the polarity switching method, spectra were acquired within the mass range of m/z 400–1300 from 0.2 to 0.6 min in the negative and from 0.8 to 1.2 min in the positive polarity mode. Phosphatidylcholine (diacyl, PC and alkyl-acyl, PC-O), phosphatidylethanolamine (diacyl, PE and alkenyl-acyl, PE-P), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG)/bis(monoacylglycero)phosphate (BMP), cardiolipin (CL), and the lyso derivatives LPC, LPE, LPI, LPS, LPG, and LCL as well as ceramide (Cer), hexosyl ceramide (HexCer), GM3 ganglioside, and sulfatide (Sulf) were detected and quantified using the negative ion mode, whereas sphingomyelin (SM), diacylglycerol (DG), triacylglycerol (TG), and cholesteryl ester (CE) were detected and quantified using the positive ion mode. In addition, PCs were also analyzed in the positive ion mode, and we detected and profiled higher brain gangliosides (GD3, GD1, GT1, and GQ1) in the negative polarity mode.
MS analyses were performed on an Orbitrap Fusion Lumos instrument (Thermo Fisher Scientific, Bremen, Germany) equipped with a robotic nanoflow ion source (TriVersa NanoMate, Advion BioSciences, Ithaca, NY, USA) using chips with a spraying nozzle diameter of 5.5 µm. The back pressure was set at 1 psi. The ionization voltages were +1.3 kV and −1.9 kV in positive and negative modes, respectively, whereas it was +1.5 kV in acquisitions with polarity switching. The temperature of the ion transfer capillary was 260 °C. Acquisitions were performed at mass resolution Rm/z 200 = 240,000 in full scan mode. In the polarity switching method, spectra were acquired within the mass range of m/z 400–1300 from 0.2 to 0.6 min in the negative and from 0.8 to 1.2 min in the positive polarity mode. Phosphatidylcholine (diacyl, PC and alkyl-acyl, PC-O), phosphatidylethanolamine (diacyl, PE and alkenyl-acyl, PE-P), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG)/bis(monoacylglycero)phosphate (BMP), cardiolipin (CL), and the lyso derivatives LPC, LPE, LPI, LPS, LPG, and LCL as well as ceramide (Cer), hexosyl ceramide (HexCer), GM3 ganglioside, and sulfatide (Sulf) were detected and quantified using the negative ion mode, whereas sphingomyelin (SM), diacylglycerol (DG), triacylglycerol (TG), and cholesteryl ester (CE) were detected and quantified using the positive ion mode. In addition, PCs were also analyzed in the positive ion mode, and we detected and profiled higher brain gangliosides (GD3, GD1, GT1, and GQ1) in the negative polarity mode.
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