Rapid screening of HGL-DTGs via ultrahigh-pressure liquid chromatography/time of flight mass spectrometry

All materials were ground in liquid nitrogen and split into aliquots of 10–100 mg fresh weight (FW), depending on the tissue. Each aliquot was extracted in 100 µL–1 mL extraction solution (80% methanol; ratio 1/10 FW/extraction solution) containing two steel balls by shaking twice at 1,200 strokes/min for 60 s using a Geno/Grinder 2000. Homogenized samples were then centrifuged at 16,000g for 20 min at 4°C. The supernatant was centrifuged again at 16,000g for 20 min at 4°C. Two independent chromatographic methods were used to resolve HGL-DTGs. Both methods used a mixture of solvent A: water with 0.1% acetonitrile and 0.05% formic acid and solvent B: acetonitrile with 0.05% formic acid. U(H)PLC was performed using a Dionex UltiMate 3000 rapid separation LC system (Thermo Fisher, http://www.thermofisher.com), combined with a Thermo Acclaim RSLC 120 C18 column (particle size 2.2 µm, average pore diameter 120Å, column dimension 2.1 × 150 mm). The gradient e steps were as follows: 0–0.5 min at 10% of B, 0.5–6.5 min up to 80% of B and 6.5–8 min at 80% of B followed by returning to the starting conditions and column equilibration. For method B, the sample gradient steps were as follows: 0–3 min at 10% B, 3–12 min up to 20% B, 12–17 min up to 35% B, 17–23 min up to 40% B, 23–25 min up to 45% B, 25–30 min up to 50% B, 30–40 min up to 90% B and 40–45 min at 90% B, followed by returning to the starting conditions and column equilibration. The injection volume was 2 µL and the flow rate 0.4 mL min⁻¹ for method A and B.

MS detection was performed using a micrOTOF-Q II, an Impact II, and a maXis UHR-Q-TOF-MS system (Bruker Daltonics) equipped with an electrospray ionization (ESI) source operating in positive ion mode. ESI conditions for the micrOTOF-Q II system were end plate offset 500 V, capillary voltage 4,500 V, capillary exit 130 V, dry temperature 180°C, and a dry gas flow of 10 L min⁻¹. ESI conditions for the Impact II UHR-Q-TOF-MS system were capillary voltage 4,500 V, end plate offset 500 V, nebulizer 2 bar, dry temperature 200°C and a dry gas flow of 8 L min⁻¹. ESI conditions for the maXis UHR-Q-TOF-MS system were capillary voltage 4,500 V, end plate offset 500 V, nebulizer 1.8 bar, dry temperature 200°C and a dry gas flow of 8 L min⁻¹. MS data were collected over a range of m/z from 100 to 1,600. Mass calibration was performed using sodium formate (50 mL isopropanol, 200 µL formic acid, 1 mL 1 M NaOH in water). Data files were calibrated using the Bruker high-precision calibration algorithm. Lock mass calibration was performed for the profiling of the stable lines using signal m/z 622.0289 (molecular formula C₁₂H₁₉F₁₂N₃O₆P₃) from the ESI Tuning Mix (Agilent Technologies, http://www.agilent.com). MS/MS experiments were performed using AutoMS/MS runs at various CID voltages from 12.5 to 22.5 eV for ammonium adducts. Instrument control, data acquisition, and reprocessing were performed using HyStar 3.1 (Bruker Daltonics). Molecular formulae were determined using SmartFormula 3D. SmartFormula calculates the elemental compositions from accurate mass as well as the isotopic pattern information using MS (SmartFormula) and MS + MS/MS information (SmartFormula 3D; Krebs and Yates, 2008; Kind and Fiehn, 2010). The mass tolerance was set to 4 mDa, and the filter H/C element ratio was set between 1 and 3. Isotope peaks were assigned using the Simulate Pattern Tool of DataAnalysis software version 4.2 (Bruker Daltonics). We used QuantAnalysis (Bruker Daltonics) to integrate the peak areas.