Waters acquity uplc xevo g2 qtof

The Latroeggtoxin-V fusion protein was cleaved by enterokinase at 25 °C overnight in a cleavage buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl, pH8.0) at an enzyme: substrate ratio of 1:50 (Sangon Biotech, Shanghai, China). The cleaved products were separated with RP-HPLC (SHIMADZU, Kyoto, Japan) using a C18 column (4.6 mm × 250 mm, 5 μm particle size, Welch Xtimate^TM, Shanghai, China). The cleaved products were eluted with a linear gradient of acetonitrile containing 0.1% TFA from 0% to 70% in 40 min at a flow rate of 1.0 mL/min. The molecular weight of the components in each elution peak was determined by electrospray ionization mass spectrometry (Waters ACQUITY UPLC/Xevo G2 QTOF, Waters, Milford, MA, USA) and thus the peak containing recombinant Latroeggtoxin-V (rLatroeggtoxin-V) was selected and lyophilized.

Triterpenoid saponin metabolite profiling was performed on a Waters ACQUITY UPLC /Xevo G2 Q-TOF (Waters Corporation, Milford, MA, USA). Samples were separated on Agilent ZORBAX Eclipse Plus C18 (2.1 mm × 50 mm, 1.8 μm). The column temperature was maintained at 35 °C, and the flow rate was 0.20 mL/min. The mobile phase consisted of acetonitrile and 0.1% formic acid. The gradient was initiated with 10% acetonitrile for 5 min, 10% acetonitrile linearly increased to 20% acetonitrile within 1 min and held at 20% for 4 min, then 20% acetonitrile increased to 30% acetonitrile within 1 min and held at 30% for 4 min, and 30% acetonitrile increased to 45% acetonitrile within 1 min and held at 45% for 4 min. The Xevo G2-S Q-TOF mass spectrometer was run in the negative mode. Mass spectra were obtained with a scanning mass range of 50 to 1500 Da. High-purity nitrogen (N₂) was used as nebulizing gas, and ultra-high pure helium (He) was used as the collision gas. Other parameters were as follows: Capillary voltage, 2.00 kV; sampling cone voltage, 40.0 V; ion source temperature, 120 °C; desolvation temperature, 350 °C; cone gas flow, 50 L/h; desolvation gas flow rate, 600 L/h; collision energy (CE), 40–60 V; and leucine enkephalin was used as lock mass (m/z 554.2615).