DiPTH-Cystine and PTH-Cysteine in Disulfide Bond Analysis Using Automated Edman Degradation.

The annotation of disulfide bridges in peptides and proteins can be an elaborate process and requires careful revision of multiple data sets to avoid wrong assignment in the structural analysis. Herein, we provide additional support to elucidate the cysteine connectivity by re-implementation of Edman sequencing for the analysis of this specific structural feature. By synthesizing diPTH-cystine and PTH-cysteine for comparison, we were able to identify the respective derivative during Edman sequencing when a disulfide bond is detected in a peptide. Application of Edman sequencing to selected peptides with two or three disulfide bridges provides further insight into the differentiation of cysteines that form a disulfide bridge for both half-cystines in the same cycle and in separated cycles. A combined approach for the implementation of automated Edman sequencing in the process of disulfide bond assignment is described to alleviate structural elucidation in the future analysis of cysteine-rich peptides and proteins.