Differentiation of frogs from two populations belonging to the Pelophylax esculentus complex by LC-MS/MS comparison of their skin peptidomes

Abstract

LC-MS/MS was applied to establish the composition of the skin peptidome of a Slovenian green frog belonging to the Pelophylax esculentus complex. As this was similar to the peptidome of the Moscow population of Pelophylax ridibundus, it allowed us to identify the Slovenian frog from the Pelophylax esculentus complex as Pelophylax ridibundus. The sequences of six new peptides from the brevinin 2 family are reported for the first time on the basis of manual interpretation of their tandem mass spectra. The structural similarity of the brevinin 2 peptides from the Moscow and Slovenian populations of Pelophylax ridibundus enables peptides from this family to be utilized as biomarkers for Pelophylax ridibundus inter- and intraspecies differentiation, and the proposed approach can be used as an analytical tool for differentiating the corresponding species and populations. The potential biological activities of the novel peptides were estimated by 2D mass mapping. The results allowed us to classify all of the available peptides belonging to the brevinin 2 family. Graphical Abstract Intraspecies identification within the green frog complex.

A New Family of Diverse Skin Peptides from the Microhylid Frog Genus Phrynomantis

“…First, their absence could be attributed to geographic differences in peptide repertoires among different P. microps populations, since this species has a wide distribution range across the savannah regions of West Africa [20,25,26]. Such variation has been found across populations of North American and European ranid frogs [60,61] and may either involve sequence differences (different populations producing different isoforms of a single peptide family) or variation in peptide expression (peptides are produced in some populations but not in others). In P. microps, such variation may be correlated to the extent to which different populations aestivate in ant nests.…”

Manual mass spectrometry de novo sequencing of the anionic host defense peptides of the Cuban Treefrog Osteopilus septentrionalis

“…36 The array of skin peptides is unique for each amphibian species and may be treated as a reliable taxonomic characteristic, 36,37 which has been used to distinguish between closely related frog species 38 and even between populations. 39 Therefore, the study of the skin peptidome represents an important tool to discover phylogenetic connection inside Anurans, allowing the evolution pathways in the amphibian kingdom and of the whole planet to be revealed. [40][41][42] Determination of the sequence of HDPs is the first step in the study of their physicochemical properties and related biological activity.…”

“…The most efficient approach so far involves recording and manual interpretation of complementary ECD/ETD, CID, and HCD spectra preferably with accurate mass measurements. [37][38][39][44][45][46] Most species of amphibians included in the family Hylidae are arboreal frogs that climb on vegetation, ranging from low grasses and shrubs to the top of the canopy, in tropical rainforests. They descend to ground level to breed and place their eggs on vegetation, tree trunks, and other substrates over water.…”

“…About 200 7 out of 8235 species of Anura inhabiting our planet have been studied, 34 and more than 1000 antimicrobial peptides, grouped in about 100 families, have been reported, 7,35 with their names being derived from the first described peptide in the family 36 . The array of skin peptides is unique for each amphibian species and may be treated as a reliable taxonomic characteristic, 36,37 which has been used to distinguish between closely related frog species 38 and even between populations 39 . Therefore, the study of the skin peptidome represents an important tool to discover phylogenetic connection inside Anurans, allowing the evolution pathways in the amphibian kingdom and of the whole planet to be revealed 40–42 …”

“…However, the amphibian peptidome is usually smaller than the transcryptome due to possible rapid peptide degradation upon secretion and uncorrelated temporal variation in transcription and peptide synthesis, 43 demonstrating at the same time a number of post‐translational modifications in the structure of the secreted peptides. The most efficient approach so far involves recording and manual interpretation of complementary ECD/ETD, CID, and HCD spectra preferably with accurate mass measurements 37–39,44–46 …”