2535 system

VK was purified and identified as described previously¹⁵ . In brief, crude venom from Vespa magnifica (Smith) was collected from Yunnan Province, China. The lyophilized crude venom was dissolved in deionized water (1 mg/mL) and filtered, then loaded onto a Sepax Bio-C18 column (21.2 × 250 mm, 10 μm). To purify VK, HPLC was put on a Waters 2535 system equipped with a manual injector and two-solvent system: (A) acetonitrile with 0.1% trifluoroacetic acid (TFA) and (B) water with 0.1% TFA. The effluent fractions corresponding to the chromatographic peak was manually collected in a tube and lyophilized for subsequent detection. Mass measurements of the separations were executed using a matrix-assisted laser desorption/ionization time of flight mass spectrometry (140,000 at 200 M/Z at a scan rate of 1.5 Hz, Q Exactive™, Thermofisher Scientific, USA). The molecular mass of the sample was determined by the reflection method with positive ion mode. The amino acids were confirmed by tandem mass spectrometry (MS/MS) and automatic amino acid analyzer. The purified component was sequenced with Edman degradation.

The lyophilized crude venom was dissolved in pure water, filtered and loaded onto a Sepax Bio-C18 column (21.2 × 250 mm, 10 μm). The HPLC was performed on a Waters 2535 system, equipped with a manual injector and two-solvent system: (A) acetonitrile with 0.1% TFA and (B) water with 0.1% TFA [18 ]. The effluent fractions corresponding to chromatographic peaks were manually collected in tubes, and lyophilized for subsequent testing. Four purified substances were collected in this way, which we designated as VMS1, VMS2, VMS3, and VMS4. Mass measurements of the isolates were performed on a matrix-assisted laser desorption/ionization time of flight mass spectrometry (5800 MALDI-TOF MS, AB Sciex, Framingham, MA, USA). The reflection method with positive ion mode was used to test the molecular mass of the samples. Tandem mass spectrometry (MS/MS) and amino acid determination by automatic amino acid analyzer, together with Edman degradation, were used to analyze the sequences of the purified components.