EThcD Benefits for the Sequencing Inside Intramolecular Disulfide Cycles of Amphibian Intact Peptides

Disulfide bonds formed by a pair of cysteine residues in the peptides' backbone represent a certain problem for their sequencing by means of mass spectrometry. As a rule, in proteomics, disulfide bonds should be cleaved before the analysis followed by some sort of chemical derivatization. That step is time-consuming and may lead to losses of minor peptides of the analyzed mixtures due to incomplete reaction, adsorption on the walls of the vials, etc. Certain problems in the de novo top-down sequencing of amphibian skin peptides are caused by the C-terminal disulfide loop, called the Rana box. Its reduction with or without subsequent derivatization was considered to be an unavoidable step before mass spectrometry. In the present study, EThcD demonstrated its efficiency in sequencing intact disulfide-containing peptides without any preliminary derivatization. Applied to the secretion of three frog species, EThcD provided the full sequence inside the intramolecular disulfide cycle for all S-S-containing peptides found in the samples, with the only exception being diarginine species. Proteolytic fragments, which are shorter than the original peptides, were helpful in some cases. HCD should be mentioned as a complementary tool to the EThcD tool, being useful as a confirmation method for some sequence details.