| MS Description
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Methanol, isopropanol, and acetonitrile of … Methanol, isopropanol, and acetonitrile of LC-MS grade were purchased from Wako. Ammonium acetate and EDTA were purchased from Wako and Dojindo, respectively. Milli-Q water was purchased from Millipore. SPLASH Lipidomics I or EquiSPLASH used as internal standards were purchased from Avanti Polar Lipids.
<LC Method>
The LC system consisted of a Waters Acquity UPLC system. Lipids were separated on an Acquity UPLC Peptide BEH C18 column (50 x 2.1 mm; 1.7 µm) (Waters, Milford, MA, USA). The column was maintained at 45°C at a flow-rate of 0.3 mL/min. The mobile phases consisted of (A) 1:1:3 (v/v/v) acetonitrile:methanol:water with ammonium formate (5 mM) and 10 nM EDTA and (B) 100% isopropanol with ammonium formate (5 mM) and 10 nM EDTA. A sample volume of 0.5-3 µL, which depended on biological samples, was used for the injection. The separation was conducted under the following gradient: 0 min 0% (B); 1 min 0% (B); 5 min 40% (B); 7.5 min 64% (B); 12 min 64% (B); 12.5 min 82.5% (B); 19 min 85% (B); 20 min 95% (B); 20.1 min 0% (B); and 25 min 0% (B). Sample temperature was maintained at 4°C.
<MS Method>
Mass spectrometric detection of lipids was performed on a quadrupole/time-of-flight mass spectrometer TripleTOF 5600 or 6600 (SCIEX, Framingham, MA, USA). All analyses were performed at the high resolution mode in MS1 (~35,000 full width at half maximum (FWHM)) and at the high sensitivity mode (~20,000 FWHM) in MS2. Data dependent MS/MS acquisition (DDA) was used. The parameters were MS1 and MS2 mass ranges, m/z 70-1250; MS1 accumulation time, 250 ms; MS2 accumulation time, 100 ms; collision energy, +40/-42 eV; collision energy spread, 15 eV; cycle time, 1300 ms; curtain gas, 30; ion source gas 1, 40(+)/50(-); ion source gas 2, 80(+)/50(-); temperature, 250°C(+)/300°C(-); ion spray voltage floating, +5.5/-4.5 kV; declustering potential, 80 V. The other DDA parameters were dependent product ion scan number, 16; intensity threshold, 100 cps; exclusion time of precursor ion, 0s; mass tolerance, 20 ppm; ignore peaks, within m/z 200; and dynamic background subtraction, True. The mass calibration was automatically performed using an APCI positive/negative calibration solution via a calibration delivery system (CDS). n via a calibration delivery system (CDS).
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