HPLC purification of Buthus martensii Karsch neurite-stimulating peptide targeting Kv channels (BmK NSPK). (A) Representative RP-HPLC chromatogram of the fraction eluted with 0.5 M NaCl in an CM-Sephadex C-50 cation ion-exchange column. The column was eluted with different percentages of solvent A (0.1% Trifluoroacetic acid (TFA) in ddH2O) and solvent B (0.085% TFA, 70% acetonitrile in ddH2O): from 0 to 5 min, 19% B, 5–20 min, 28% B; 20–55 min, 48% B; 55–60 min, 19% B at a flow rate of 2 mL/min. The arrowhead indicates the peak of BmK NSPK. (B) Representative RP-HPLC chromatogram of purified BmK NSPK eluted with gradient of acetonitrile: solvent A (0.1% TFA in ddH2O) and solvent B (0.085% TFA, 70% acetonitrile in ddH2O): from 0 to 10 min, 99.5% B; 10–70 min, 58% B; 70–75 min, 99.5% B at a flow rate of 1 mL/min. A single peak (indicated by arrowhead) was observed, suggesting that BmK NSPK is of high purity. (C) ESI-MS of BmK NSPK. The multiple ion charges of 1321.7, 991.6, and 793.5 m/z corresponded to [M+3H]3+, [M+4H]4+, and [M+5H]5+, respectively.