Waters 2795 hplc system

The crude venom of C. longipes was obtained by electrical stimulation, freeze-dried, and stored at −80 °C until further use. Lyophilized venom was dissolved in double-distilled water (ddH₂O) and purified by both reverse-phase (RP-HPLC) and ion-exchange chromatography. RP-HPLC was performed using a C18 column (10 × 250 mm, 5 μm; Welch Materials Inc., Shanghai, China) on an analytical Waters 2795 HPLC system. The acetonitrile gradient increased at a rate of 1% per minute from 5–50%, using a flow rate of 3 mL/min. Eluted fractions were lyophilized and further fractionated using ion-exchange chromatography with an XB-SCX column (4.6 mm × 250 mm, 5 μm; Welch, China) on a preparative Hanbon HPLC system. The NaCl gradient increased at a rate of 2% per minute from 0–70% at a flow rate of 1 mL/min. Collected fractions containing Cl6a and Cl6b were subjected to further desalination by RP-HPLC using a C18 column (4.6 mm × 250 mm, 5 μm; Welch, China) on an analytical Waters 2795 HPLC system. In the second round of RP-HPLC, the acetonitrile gradient increased from 27–33% at a rate of 0.5% per minute and a flow rate of 1 mL/min. For C18 RP-HPLC, solvent A was 0.1% trifluoroacetic acid (TFA) in water and solvent B was 0.1% TFA in acetonitrile. Cl6a- and Cl6b-containing fractions were lyophilized and stored at −20 °C until further use.

μ-SPRTX-Hv2 was synthesized by using a Fmoc (N-(9-fluorenyl)methoxycarbonyl)/tert-butyl strategy and HOBt/TBTU/NMM coupling method [44 (link)]. The refolding buffer contains (in mM): 100 NaCl, 5 GSH, 0.5 GSSG, and 100 Tris (pH = 7.4, adjusted with HCl). The linear peptide was diluted with the refolding buffer to a final concentration of 0.01 mg/mL. The solution was stirred slowly at room temperature for 24 h and the refolding reaction was monitored by MALDI-TOF MS analysis. The reaction was terminated by adding TFA to a final concentration of 0.2%, and the reaction mix was subjected to RP-HPLC purification (C18 column, 4.6 × 250 mm, 5 μm, Welch Materials Inc., Shanghai, China) using a 35-min linear acetonitrile gradient from 25% to 46% at 1 mL/min flow rate. The co-elution experiments were performed in Waters 2795 HPLC system equipped with Water 2487 detector (Waters Corporation, Milford, MA, USA) by using a 25-min linear acetonitrile gradient from 20% to 45% at 1 mL/min flow rate (C18 column, 4.6 × 250 mm, 5 μm, Welch Materials Inc., Shanghai, China).

κ-LhTx-1 linear peptide was synthesized using a Fmoc [N-(9-fluorenyl)methoxycarbonyl]/tert-butyl strategy and HOBt/TBTU/NMM coupling method. The produced linear peptide was poorly dissolved in the basic refolding solution [5 mM GSH, 0.5 mM GSSG, 100 mM NaCl, 0.1 M Tris-HCl (pH = 7.4)], therefore 4 M guanidine hydrochloride was added to improve its solubility. Peptide was refolded at a concentration of 0.1 mg/ml, after 5 h stir at 4°C, the guanidine hydrochloride concentration in the refolding mix was sequentially diluted to 3, 2, and 1 M with basic refolding solution (one dilution per 5 h). RP-HPLC and MALDI-TOF MS analysis was used to monitor the refolding process. At last, the refolding reaction was terminated by adding TFA to a final concentration of 0.2%, and the mix was subjected to RP-HPLC purification to collect the correctly refolded toxin (C18 column, 10 × 250 mm, 5 μm, Welch Materials Inc., Shanghai, China; a 45-min linear acetonitrile gradient from 5 to 50% was used, the flow rate is 3 ml/min). The correct refolding of synthesized κ-LhTx-1 was also confirmed by its co-elution with the native κ-LhTx-1 in RP-HPLC analysis (C18 column, 4.6 × 250 mm, 5 μm, Welch Materials Inc., Shanghai, China) using a 50-min linear acetonitrile gradient from 5 to 55% at 1 ml/min flow rate (Waters 2795 HPLC system, Waters Corporation, Milford, MA, United States).