Peptaibol Analysis Using HPLC-ESI-MS

The crude peptaibol extracts were measured by using an HPLC-ESI-MS instrument with an Agilent 1100 system (Agilent Technologies, Palo Alto, CA, USA) controlled by a ChemStation software (A09.03; Agilent Technologies, Palo Alto, CA, USA). The system was equipped with a binary pump, a vacuum degasser, a µWell-plate autosampler, as well as a Jones Model 7990 Space column heater (Jones Chromatography Ltd., Lakewood, CO, USA). Peptaibol separation was carried out on Gemini NX-C18 HPLC column (150 mm × 2.0 mm, 3 µm; Phenomenex Inc., Torrance, CA, USA). Solvent A was H₂O with 0.05% (v/v) trifluoroacetic acid (TFA), while Solvent B was acetonitrile/methanol 1/1 (v/v) with 0.05% (v/v) TFA. The flow rate was set to 0.2 mL min⁻¹, the gradient program for Solvent B to 65%—0 min, 65%—5 min, 80%—45 min, 100%—70 min, 100%—75 min, 65%—76 min, 65%—81 min, the column temperature to 40 °C, and the injection volume to 5 µL. The ESI-IT-MS instrument was Varian 500 MS (Agilent Technologies, Palo Alto, CA, USA) with ESI source in positive mode at normal scan speed and controlled by the 500-MS Mass Spec module driver of the Varian Workstation software (6.6/SP1; Varian Inc., Palo Alto, CA, USA). ESI parameters were set to the following values: spray chamber temperature: 50 °C, drying gas (N₂) pressure: 30 psi, drying gas temperature: 350 °C, nebuliser gas (N₂) pressure: 50 psi, needle voltage: 5704 V, spray shield voltage: 600 V. The general parameters were set as the maximum scan times at 2.78, 2 μScans averaged, data rate at 0.36 Hz and multiplier offset at 0. The ionization control parameters were set as target TIC wet at 100% and max ion time at 250,000 μsec, scan parameters as capillary voltage was set at 66 V, RF loading at 147%, while the MS scan parameters were set as low mass m/z at 100, high mass m/z at 2000. The MS² measurements of selected y₇ fragments were carried out with the following excitation storage level (m/z)/excitation amplitude (V) conditions: m/z 754.5 (204.5/2.95), m/z 755.5 (204.8/2.96) m/z 768.5 (208.0/3.00), and m/z 769.5 (208.3/3.00).
Based on a calibration with alamethicin standard (Sigma-Aldrich Ltd., Budapest, Hungary), the peptaibol contents of the crude extracts were also calculated.