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The liquid chromatography system consisted … The liquid chromatography system consisted of a Waters Acquity UPLC system (Waters Inc.). Mobile phase A was 1:1:3 acetonitrile:methanol:water (v/v/v) with 5 mM ammonium acetate and 10 nM EDTA. Mobile phase B was 100% isopropanol with 5 mM ammonium acetate and 10 nM EDTA. The LC column was an Acquity UPLC Peptide BEH C18 column (50×2.1 mm; 1.7 μm; 130Å). The gradient was 0 min, 0% B; 1 min, 0% B; 5 min, 40% B; 7.5 min, 64% B; 12.0 min, 64% B; 12.5 min, 82.5% B, 19 min, 85% B; 20 min, 95% B; 20.1 min, 0% B; 25 min, 0% B. The column flow rate was 0.3 mL/min, the autosampler temperature was 5°C, the injection volume was 1 uL for mouse liver tissue and 2 μL for the other samples. The column temperature was 45°C.
MS was performed on an AB Sciex TripleTOF 5600+ system (Q-TOF) equipped with a DuoSpray ion source. All analyses were performed in the high sensitivity mode for both TOF-MS and product ion scanning. Mass calibration was automatically performed every five injections using an APCI positive/negative calibration solution and a calibration delivery system (CDS). Data-dependent MS/MS acquisition (DDA) was used. The common parameters in both positive and negative ion mode were collision energy, 45 V; collision energy spread, 15 V; mass range, m/z 70-1250; curtain gas, 30; ion source gas 1, 50; ion source gas 2, 50; temperature, 500°C for mouse ear tissue and 300°C for the other samples; declustering potential, 80 V; RF transmission, default. The ion spray voltage floating in positive/negative ion mode was +5.5/–4.5 kV, respectively. The DDA parameters in both positive and negative ion mode were MS1 accumulation time, 250 ms; MS2 accumulation time, 100 ms; cycle time, 1,300 ms; dependent product ion scan number, 10; intensity threshold, 100; exclusion time of precursor ion, 5 sec; mass tolerance, 20 mDa; ignore peaks, within 6 Da; dynamic background subtraction, TRUE. Da; dynamic background subtraction, TRUE.
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