| MS Description
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Ultrapure water was produced with a Milli- … Ultrapure water was produced with a Milli-Q Integral system equipped with a LC-Pak Polisher and a 0.22 μm point of use membrane filter cartridge (EMD Millipore, Billerica, MA, USA). Ammonium fluoride and LC/MS grade ammonium acetate were purchased from Millipore Sigma (St. Louis, MO, USA). Isopropanol of LC/MS grade “LiChrosolv” was purchased from Sigma Aldrich. HPLC-grade acetonitrile and methanol were purchased from Honeywell (Morristown, NJ, USA). SPLASH lipidomics I used as internal standards were purchased from Avanti Polar Lipids. Chloroform was purchased from Honeywell (Charlotte, North Carolina).
<LC Method>
The LC system consisted of an Agilent 1290 Infinity II (Agilent Technologies, Santa Clara, CA, USA) with a pump, a column oven and an autosampler. Lipids were separated on an Agilent InfinityLab Poroshell 120 EC-C18 column (100 x 3.0 mm; 2.7 µm) coupled to an Agilent InfinityLab Poroshell 120 EC-C18 column precolumn (5 x 3.0 mm; 2.7 µm). The column was maintained at 50°C at a flow-rate of 0.6 mL/min. The mobile phases consisted of (A) 9:1 (v/v) water:methanol with ammonium acetate (10 mM) and ammonium fluoride (0.2 mM) and (B) 2:3:5 (v/v/v) acetonitrile:methanol/isopropanol with ammonium formate (10 mM) and ammonium fluoride (0.2 mM). A sample volume of 5 µL was used for tinjections in ESI(+) and ESI(-). Separation was conducted under the following gradient: 0 min 70% (B); 1 min 70% (B); 3.5 min 86% (B); 10.0 min 86% (B); 11.0 min 100% (B); 17.0 min 100% (B); 17.1 min 70% (B); 19.0 min 70% (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 6546 Q-TOF (Agilent Technologies, Santa Clara, CA, USA). Simultaneous MS1 and MS/MS (data-dependent MS/MS) acquisition was used. The parameters were as follows: gas temperature, 200°C; gas flow, 10 L/min; nebulizer (psig), 50; sheath gas temperature, 300°C; sheath gas flow, 12 L/min; VCap, 3.5 kV and -3.0 kV for ESI(+) and ESI(-), respectively; nozzle voltage, 0 V; fragmentor, 150 V; skimmer, 65 V; octupole RF Vpp, 750 V; MS1 and MS2 mass range, m/z 40-1700; min MS and MS/MS acquisition rate: 3 spectra/s; isolation width, narrow (~1.3 m/z); max precursors per cycle, 3; precursor abundance-based scan speed, target 25,000 counts/spectrum; use MS/MS accumulation time limit, yes; reject precursors that cannot reach target TIC, no; threshold for MS/MS, 5,000 counts and 0.001%; active exclusion enabled, one repeat, then exclude for 0.05 minutes; purity, stringency 70%, cut off 0%; isotope model, common organic molecules; sort precursors, 1,2,unknown; static exclusion ranges, m/z 40 to 151 for ESI(+) and m/z 40 to 210 for ESI(-); collision energy, 20 eV for ESI(+) and 25 eV for ESI(-). The instrument was tuned using a reference mass m/z 121.050873, m/z 1221.990637 (+) and m/z 119.03632, m/z 980.016375 (-). (+) and m/z 119.03632, m/z 980.016375 (-).
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