Dual agilent jet stream esi

To establish the phytochemical compositions of JM1–JM6, LC–PDA–ESI–MS/TOF analysis was performed on a 1260 Infinity chromatography system hyphenated to a 6230 TOF mass spectrometer and Dual Agilent Jet Stream ESI (Agilent Technologies, Santa Clara, CA, USA). The MS conditions were as follows: electrospray ionization (ESI) source in both positive and negative ionization mode, a gas flow of 12 L/min, a gas temperature of 325 °C, a nebulizer pressure of 45 psi, and capillary voltages of 4500 and 2500 V for positive and negative ion modes, respectively. The analysis was performed using a Kinetex XB-C18 column (150 × 2.1 mm, 1.7 µm; Phenomenex, Torrance, CA, USA). The mobile phases were UPW (solvent A) and MeCN (solvent B), both with 0.1% HCOOH. The gradient started with the elution of 5% solvent B over 1.5 min. Then, within 22 min, the concentration of solvent B reached 28%, in 35 min it reached 75%, and finally in 45 min it reached 95%, with a linear gradient. After 3 min of maintaining the 95% B concentration, in 49 min, the system returned to its initial conditions, and conditioning continued for 6 min. The total run time of the analysis was 55 min at 25 °C. The injection volume was 1.0 µL, and the flow rate was 0.1 mL/min.

The purity at the target residue was determined by quantitative MS. The ELP-GFP proteins were buffer exchanged and diluted to 15 µM in digestion buffer (50 mM Tris, pH 8.0, and 0.5 mM CaCl₂), and were digested with 1.5 µM thermolysin (Promega) for 6 h at 80 °C. The resulting ELP-peptides were quantified using standard curves based on synthetic peptides (SI Appendix, Fig. S2). High-resolution MS data were collected using an Agilent iFunnel 6550 quadrupole time-of-flight (TOF) MS with an electrospray ionization (ESI) source, coupled to an Agilent Infinity 1290 ultrahigh-performance liquid chromatography system with an Agilent Eclipse Plus C18 1.8 μm, 4.6 × 50-mm column. Solvents used were (solvent A) water 0.1% formic acid and (solvent B) CH₃CN 0.1% formic acid. Mass spectra were gathered using Dual Agilent Jet Stream ESI in positive mode. The mass range was set from 110 to 1,700 m/z with a scan speed of three scans per second. The capillary and nozzle voltages were set to 5,500 and 2,000 V, respectively. The source parameters were set with a gas temperature of 280 °C and a flowrate of 11 liters/min, nebulizer at 40 psig, and sheath gas temperature at 350 °C at a flow of 11 liters/min. MS data were acquired with MassHunter Workstation Data Acquisition (version B.06.01, Agilent Technologies) and analyzed using MassHunterQualitative Analysis (version B.07.00, Agilent Technologies).