Direct assignment of disulfide bonds by Edman degradation of selected peptide fragments

Abstract

Disulfide linkages in peptides or proteins were analyzed by automated gas-phase Edman sequencing performed in minimized reducing agents. If cystine linkage was regulated at the same position in two peptides during peptide preparation, the diphenylthiohydantoin derivative of cystine was significantly recovered by Edman reaction. In contrast, when the crosslinked half cystines were present at different positions in the peptides, the derivative could be poorly detected. Upon direct sequence analysis of intact bovine insulin, the PTH derivatives of cystine from both Cys-A7 and Cys-B7 were significantly released after Edman cycle 7 and gave approximately 20% recovery of common PTH amino acids. However, Cys-A11 linked to Cys-A6 was poorly detectable after Edman cycle 11. For general use of this method, proteins need to be subjected to several sets of proteolytic or chemical cleavages in the hope that at least one of the fragments will have cystine linkage at the same position. This method was applied to several fragments of platelet-derived growth factor B chain and brain-derived neurotrophic factor.