| MS Description
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The liquid chromatography system consisted … The liquid chromatography system consisted of an Agilent 1290 system (Agilent Technologies Inc.) with a pump (G4220A), a column oven (G1316C), and an autosampler (G4226A). For hydrophilic metabolite analysis, mobile phase A was 10 mM ammonium formate with 0.125 % formic acid in water; mobile phase B was 95:5 acetonitrile:water (v/v) with 10 mM ammonium formate and 0.125 % formic acid. An Acquity UPLC BEH Amide column (150×2.1 mm; 1.7 µm) coupled to a VanGuard BEH Amide pre-column (5×2.1 mm; 1.7 µm) (Waters; Milford, MA, USA) was used. The gradient was 0 min, 100% B; 2 min, 100% B; 7.7 min, 70% B; 9.5 min, 40% B; 10.3 min, 30% B; 12.8 min, 100% B; 16.8 min, 100% B. The column flow rate was 0.4 mL/min, autosampler temperature was 4 °C, injection volume was 2 µL, and column temperature was 45 °C. For lipid analysis, mobile phase A was 60:40 acetonitrile:water (v/v) with 10 mM ammonium formate and 0.1% formic acid; mobile phase B was 90:10 isopropanol:acetonitrile (v/v) with 10 mM ammonium formate and 0.1% formic acid.
The lipidomic LC method utilized an Acquity UPLC charged-surface hybrid (CSH) C18 column (100×2.1 mm; 1.7 µm) coupled to an Acquity CSH C18 VanGuard pre-column (5×2.1 mm; 1.7 µm) (Waters; Milford, MA, USA). The gradient was 0 min, 15% B; 2 min, 30% B; 2.5 min, 48% B; 11 min, 82% B, 11.5 min, 99% B; 12 min, 99% B; 12.1 min, 15% B; 15 min, 15% B. The column flow rate was 0.6 mL/min, autosampler temperature was 4 °C, injection volume was 3 µL in positive mode and 5 µL in negative mode, and column temperature was 65 °C.
Mass spectrometry was performed on an AB Sciex TripleTOF 5600+ system (Q-TOF) equipped with a DuoSpray ion source. All analyses were performed at the high sensitivity mode for both TOF MS and product ion scan. The mass calibration was automatically performed every 10 injections using an APCI positive/negative calibration solution via a calibration delivery system (CDS). For hydrophilic interaction chromatography analysis, SWATH (sequential window acquisition of all theoretical mass spectra) acquisition with positive ion mode was used as the data independent acquisition system. The SWATH parameters were MS1 accumulation time, 50 ms; MS2 accumulation time, 30 ms; collision energy, 45 V; collision energy spread, 15 V; cycle time, 640 ms; Q1 window, 25 Da; mass range, m/z 50–500. The other parameters were curtain gas, 35; ion source gas 1, 50; ion source gas 2, 50; temperature, 300 °C; ion spray voltage floating, 4.5 kV; declustering potential, 100 V; RF transmission, m/z 40: 33%, m/z 120: 33%, and m/z 390: 34%. For lipid analysis, six different methods were used; DDA (data-dependent acquisition) with positive ion mode, DDA with negative ion mode, SWATH acquisition (Q1 window, 21 Da) with positive ion mode, SWATH acquisition (Q1 window, 21 Da) with negative ion mode, SWATH acquisition (Q1 window, 65 Da) with positive ion mode, and SWATH acquisition (Q1 window, 65 Da). The common parameters in both SWATH/DDA and positive/negative ion mode were collision energy, 45 V; collision energy spread, 15 V; mass range, m/z 100–1250; curtain gas, 35; ion source gas 1, 60; ion source gas 2, 60; temperature, 350 °C; declustering potential, 80 V; RF transmission, m/z 80: 50%, m/z 200: 50%. The ion spray voltage floating of positive/negative ion mode were +5.5/–4.5 kV, respectively. The DDA parameters in both positive and negative ion modes were MS1 accumulation time, 100 ms; MS2 accumulation time, 50 ms; cycle time, 650 ms; dependent product ion scan number, 10; intensity threshold, 500; exclusion time of precursor ion, 5 s; mass tolerance, 20 mDa; ignore peaks, within 6 Da; dynamic background subtraction, TRUE. The SWATH parameters of 21/65 Da Q1 window were MS1 accumulation time, 100/50 ms; MS2 accumulation time, 10/30 ms; cycle time, 731/640 ms; Q1 window, 21/65 Da. cle time, 731/640 ms; Q1 window, 21/65 Da.
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