Disulfide bonds are one of major post-translational modifications for protein skeleton construction. Correct folding featuring appropriate disulfide bonds is especially of great concern because it can directly affect the activity and stability of venom proteins. An efficient method is required for disulfide bond investigation not only to optimize the folding condition but also to assure the quality of manufactured biologics. In this study, a welldeveloped platform using mass spectrometry was applied to profile the disulfide linkages of a synthetic toxin, cardiotoxin A3 (composed of 60 amino acid residues, with four disulfide bonds), during the refolding process. The bio-active cardiotoxin A3 product with correct folding was obtained by using an optimized dilution refolding method with the presence of GSSG/GSH in weak alkaline conditions. Based on the MS data, we were able to determine the protein fraction that performs exact disulfide linkage patterns as those found in native CTXA3, and optimize the manufacturing condition to generate venom with matched DS patterns. The further bioassays proved that the synthetic peptide product exhibited similar biochemical properties and cell penetration activity as the native one. The study results indicate that bioactive peptides with correct disulfide linkages could be obtained from a solid phase peptide synthesis (SPPS) approach which can be applied in the pharmaceutical industry to fulfill the requested amount for novel drug development. Eventually, an MS-based disulfide analysis platform can provide significant information for protein structure-function studies, and be the examination tool for cystein-rich bioproduct qualification.
