Combination of Capillary Electrophoresis and Ion mobility coupled to Mass Spectrometry and Theoretical Calculations for cysteine connectivity identification in peptides bearing two intra-molecular disulfide bonds

Disulfide bonds are post translational modification playing essential roles in the biological activity and stability of numerous peptides and proteins. Intra-molecular disulfide bonds are found in various natural-occurring peptides such as animal venoms. In such peptides, the appropriate cysteine connectivity provides the required conformation for efficient binding to their molecular targets, which ensures their bioactivity. The characterization of cysteine pairing is still a challenging issue in the analysis of peptides targeting pharmaceutical or pharmacological utilizations. Thus, the use of sensitive, robust and efficient characterization techniques to access the cysteine pairing is crucial.
In our workflow,the separation of the disulfide isomers of three peptides bearing two intra-molecular disulfide bonds but different cysteine connectivities have been tested using Capillary Zone Electrophoresis (CZE) and Ion Mobility (IM) coupled to Mass Spectrometry (MS). Results show that CZE-MS and IM-MS act as complementary techniques to unambiguously determine the cysteine connectivity of a given peptide. Indeed, the combination of the relative migration time to a reference peptide in CZE-MS, the drift time in IM-MS and the generation of fragments by Collision Induced Dissociation (CID) led to the attribution of the disulfide connectivities in all studied cases. Finally, theoretical calculations were performed to model the different structures in gas phase and solution, supporting the experimental observations on the basis of the predicted physicochemical properties.