HNTX-XXI, a peptide toxin derived from the venom of the spider Ornithoctonus hainana, comprises a 64-amino-acid protein architecture that notably incorporates eight cysteine residues positioned at positions 2, 10, 14, 16, 17, 23, 36, and 63. The close spatial proximity of Cys16 and Cys17 poses a challenge in resolving their disulfide bridge configurations using standard methodologies. In this study, we introduce an innovative and highly efficient approach for delineating disulfide pairings in peptides containing adjacent cysteines. Our methodology integrates a two-step proteolytic digestion strategy utilizing trypsin and Glu-specific staphylococcal V8 protease coupled with a subsequent round of Edman degradation. This multifaceted approach enables the precise characterization of the disulfide bonds within the peptide. Specifically, targeted proteolysis by trypsin and V8, followed by reversed-phase HPLC separation of the resulting peptides, facilitated the unambiguous identification of disulfide linkages between Cys10-Cys23 and Cys14-Cys63. For the fragment containing the four remaining cysteines, a single cycle of Edman degradation was employed, strategically breaking the peptide bond between the adjacent cysteines. This pivotal step enabled the isolation and analysis of the resulting fragments. Subsequently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was utilized, revealing the presence of two additional disulfide bonds: Cys2-Cys17 and Cys16-Cys36. Collectively, these findings allow for the definitive assignment of the four disulfide linkages in HNTX-XXI as Cys2-Cys17, Cys10-Cys23, Cys14-Cys63, and Cys16-Cys36. This rapid and sensitive methodology represents a significant advancement in the structural characterization of peptide toxins with complex disulfide bond patterns, underscoring its potential for broad application in the field of venom peptide research.
