Mass spectrometry

The HPLC-ESI-MS measurements were carried out by the Northwest Metabolomics Research Center at the University of Washington using an Agilent 6545 Q-TOF mass spectrometer coupled with an Agilent 1290 Infinity LC pump, and an Agilent 6520 Q-TOF mass spectrometer coupled with Agilent 1260 Infinity LC system (Agilent Technologies). Samples consisted of phenol-chloroform extracts (prepared as described above) of either Buffer A alone or 50 μM purified CST in Buffer A. The HPLC separation was performed using a Waters XBridge BEH Amide column (15 cm x 2.1 mm, 2.5 μm). The mobile phase consisted of (A) H₂O:acetonitrile (95:5, v/v), 5 mM ammonium acetate, and 0.1% acetic acid, and (B) H₂O:acetonitrile (5:95, v/v), 5 mM ammonium acetate, and 0.1% acetic acid. Gradient operation was initiated at 94% of solvent B, and it decreased to 78% at t = 6.5 min, and to 39% at t = 12.0 min. Composition was maintained at 39% of solvent B until t = 18.5 min, followed by an increase to 94% at t = 19.0 min, and maintained at this condition until t = 35.0 min (total experimental time for each injection). The flow rate was 0.3 ml/min, the injection volume was 5 μl, followed by H₂O:acetonitrile (5:95, v/v) needle wash for 10 s. The column was maintained at 35°C. The ESI conditions were as follows: electrospray ion source ESI Agilent Jet Stream Technology in positive ionization mode; voltage 3.8 kV; desolvation temperature 325°C; cone flow 20 l/h; desolvation gas flow 600 l/h; nebulizer pressure 45 psi, N₂ was used as drying gas; MS scan rate of 5 spectra/s across the range m/z 60–1000, threshold count: 200; MS/MS acquisition rate 3 spectra/s, targeting precursor ion m/z 338.0701. Isolation width of 1.3 m/z, collision energies of 10 and 15 eV, m/z range of 50–500. Data were acquired using MassHunter Data Acquisition Workstation v. B.06.01.6157 software (Agilent Technologies, Palo Alto, CA). All solvents were LC-MS grade (Fisher Scientific). DI water (18.2 MΩ•cm at 25°C) was provided in-house by a Synergy Ultrapure Water System (EMD Millipore).