LC-MS/MS with 2D mass mapping of skin secretions' peptides as a reliable tool for interspecies identification inside Rana esculenta complex.

Identification of species constituting Rana esculenta complex represents a certain problem as two parental species Rana ridibunda and Rana lessonae form their hybrid R. esculenta, while external signs and sizes of the members of this complex are intersected. However the composition of skin secretion consisting mainly of peptides is different for the species of the complex. LC-MS/MS is an ideal analytical tool for the quantitative and qualitative analysis of these peptides. The results covering elemental composition of these peptides, their levels in the secretion, as well as their belonging to a certain family of peptides may be visualized by means of 2D mass maps. The proposed approach proved itself to be a perspective tool for the reliable identification of all 3 species constituting R. esculenta complex. Easy distinguishing between the species may be achieved using 2D maps as fingerprints. Besides this approach may be used to study hybridogenesis and mechanisms of hemiclonal transfer of genetic information, when rapid and reliable identification of species involved in the process is required.

De novo sequencing of peptides secreted by the skin glands of the Caucasian Green Frog Rana ridibunda.

Amphibian skin glands are known to secrete various types of bioactive peptides. The array of these peptides is specific for every frog species. The present research deals with the identification of peptides isolated from the skin secretion of the Marsh frog R. ridibunda inhabiting the Kolkhida Canyon of the Caucasian region. The research is based on comprehensive high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) analysis of intact and chemically modified peptides. In particular, an oxidation procedure was applied directly to the crude skin secretion to open S--S loops whereas N-terminal acetylation was additionally carried out for one individual peptide. Sequences were determined by manual interpretation of electron capture dissociation (ECD) and collisionally induced dissociation (CID) tandem mass spectra. A total of 29 peptides were identified in the skin secretion of the Caucasian Marsh frog. The peptide profile is represented with disulfide-containing peptides belonging to the brevinin, esculentin and ranatuerin families, neuropeptides of the bradykinin and bombesin families. Two identified peptides belonging to the ranatuerins are the first peptides of this family discovered in the skin secretions of European frogs. Ten of the identified peptides coincide with those reported earlier for the European Edible frog. Another ten are identical to those found in R. ridubunda from the Moscow region. This fact verifies the described method as being an efficient analytical tool to compare intra- and interspecific variabilities.