| MS Description
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Methanol, isopropanol, and acetonitrile of … Methanol, isopropanol, and acetonitrile of LC-MS grade were purchased from Wako. Ammonium formate and formic acid were purchased from Wako. Water was purchased from Millipore.
<LC Method>
The LC system consisted of a Bruker Elute UHPLC system. Lipids were separated on an Acquity UPLC HSS T3 C18 column (50 x 1.0 mm; 1.8 µm) (Waters, Milford, MA, USA). The column was maintained at 55°C at a flow-rate of 0.15 mL/min. The mobile phases consisted of (A) 200:800:10:1 (v/v/v/v) acetonitrile:water:1M ammonium formate:formic acid and (B) 100:900:10:1 (v/v/v/v) acetonitrile:isopropanol:1M ammonium formate:formic acid. A sample volume of 2 µL was used for the injection. The separation was conducted under the following gradient: 0 min 35% (B); 3 min 70% (B); 7 min 85% (B); 10 min 90% (B); 12 min 90% (B); 12.5 min 35% (B); and 15 min 35% (B). Sample temperature was maintained at 10°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). The PASEF mode acquisition, i.e. data dependent MS/MS acquisition with the ion mobility separation, was performed by the following parameters. The parameters were MS1 mass ranges, m/z 200-1600 and MS2 mass ranges, m/z 50-1600; MS1 and MS2 accumulation time, 148.5 ms; mobility range 0.55-1.9; collision energy 30 eV; end plate offset, 500 V; capillary voltage, +4.2/-4.2 kV; nebulizer pressure, 2 bar; dry gas, 8.0 l/min; dry temperature, 200°C; funnel 1 RF (radio frequency), 300 Vpp; funnel 2 RF, 250 Vpp; multipole RF, 200 Vpp; deflection delta, 70 V; quadrupole ion energy, 5 eV; collision transfer energy, 15 eV; collision RF, 1100 Vpp; transfer time 54 µs; pre-pulse storage, 5 µs; tims transfer Δ1, -20 V/+20 V; tims transfer Δ2, -120 V/+120 V; tims transfer Δ3, +70 V/-70 V; tims transfer Δ4, +100 V/-100 V; tims transfer Δ5, 0 V; tims transfer Δ6, +100 V/-100 V; intensity threshold, 2500 cts; target intensity, 20000 cts; exclusion time of precursor ion, 0.1 min. The mass calibration was automatically performed using 5 mM sodium formate calibration solution. 5 mM sodium formate calibration solution.
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