| 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.
<LC Method>
The LC system consisted of a Bruker Elute UHPLC 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 hybrid trapped ion mobility-quadrupole time-of-flight mass spectrometer (timsTOF Pro, Bruker Daltonics, Bremen, Germany). Data dependent MS/MS acquisition (DDA) was used. The parameters were MS1 mass ranges, m/z 200-2500 and MS2 mass ranges, m/z 50-2500; MS1 cycle time, 0.5 sec; MS2 accumulation 14 Hz ; collision energy, 30 eV; end plate offset, 500 V; capillary voltage, +4.5 kV/-4.2 kV; nebulizer pressure, 2 bar; dry gas, 10.0 l/min; dry temperature, 220°C/200°C; funnel 1 RF, 300 Vpp; funnel 2 RF, 200 Vpp/250 Vpp; multipole RF, 200 Vpp; deflection delta, +70 V/-70 V; quadrupole ion energy, 6 eV/5 eV; collision transfer energy, 14 eV/15 eV; collision RF, 1100 to 1800 Vpp stepping; transfer time 45 to 75 µs stepping; pre-pulse storage, 10 µs/5 µs; intensity threshold, 31cts.; exclusion time of precursor ion, 0.2 min. The mass calibration was automatically performed using 5 mM sodium acetate calibration solution. 5 mM sodium acetate calibration solution.
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