Abstract
Amphibians, as one of the leaders of immune resistance, have lived on Earth for hundreds of millions of years. Their dorsal glands produce a cocktail of biologically active peptides that successfully fight microorganisms and even predators. Since this mechanism prevents the development of pathogen resistance, antimicrobial peptides are very promising pharmaceuticals for future generations. Mass spectrometry is the most powerful tool for sequencing peptides/proteins. For over 30 years of studies in this field, mass spectrometry has resolved all the problems associated with the de novo sequencing of amphibian peptides. This review covers the modern de novo sequencing algorithms that enable achieving complete sequence coverage of all frog peptides, including long ones (up to 46 amino acids). Accurate mass measurements have reliably solved the problem of isobaric amino acids. Moreover, there is no longer any need to carry out any preliminary derivatization procedures such as breaking disulfide bonds or N-terminal acetylation. EThcD and ExD tools with manual spectra interpretation provide an efficient approach for reliable differentiation between isomeric leucine and isoleucine residues in the chain, using secondary w- and d-ions, and they resolve the problems of sequencing inside the intact S-S cycles.