Combination of Capillary Zone Electrophoresis-Mass Spectrometry, Ion Mobility-Mass Spectrometry, and Theoretical Calculations for Cysteine Connectivity Identification in Peptides Bearing Two Intramolecular Disulfide Bonds

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Abstract
Disulfide bonds between cysteine residues are commonly involved in the stability of numerous peptides and proteins and are crucial for providing biological activities. In such peptides, the appropriate cysteine connectivity ensures the appropriate conformation allowing an efficient binding to their molecular targets. Disulfide bond connectivity characterization is still challenging and is a critical issue in the analysis of structured peptides/proteins targeting pharmaceutical or pharmacological utilizations. This study describes the development of new and fast gas-phase and in-solution electrophoretic methods coupled to mass spectrometry to characterize the connectivity of the disulfide bond. For this purpose, disulfide isomers of three peptides bearing two intra-molecular disulfide bonds but different connections between cysteines have been investigated. Capillary Zone Electrophoresis (CZE) and Ion Mobility (IM) both coupled to Mass Spectrometry (MS) were used to perform the separation in both aqueous and gas phases, respectively. The ability of each technique to separate these isomers has been critically evaluated and compared. Finally, theoretical calculations were performed to support and explain the experimental data based on their predicted physicochemical properties.
Funding Information
  • FP7 Health
  • F?d?ration Wallonie-Bruxelles
  • European Regional Development Fund
  • Fonds De La Recherche Scientifique - FNRS

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