Differentiation of Central Slovenian and Moscow populations of Rana temporaria frogs using peptide biomarkers of temporins family.

Skin secretion represents the only means of defense for the majority of frog species. That phenomenon is based on the fact that the main components of the secretion are peptides demonstrating greatly varying types of bioactivity. They fulfill regulatory functions, fight microorganisms and may be even helpful against predators. These peptides are considered to be rather promising pharmaceuticals of future generation as according to the present knowledge microorganisms are unlikely to develop resistance to them. Mass spectrometry sequencing of these peptides is the most efficient first step of their study providing reliably their primary structures, i.e., amino acids sequence and S-S bond motif. Besides discovering new bioactive peptides, mass spectrometry appears to be an efficient tool of taxonomy studies, allowing for distinguishing not only between closely related species, but also between populations of the same species. Application of several tandem mass spectrometry tools (CID, HCD, ETD, EThcD) available with Orbitrap mass analyzer allowed us to obtain full sequence of about 60 peptides in the secretion of Slovenian population of brown ranid frog Rana temporaria. The problem of sequence inside C-terminal cycle formed by two Cys and differentiation of isomeric Leu and Ile residues was done in top-down mode without any derivatization steps. Besides general biomarkers of Rana temporaria species, Central Slovenian population of Rana temporaria demonstrates six novel temporins and one brevinin 1, which may be treated as biomarkers of that population.

Composition and antimicrobial activity of the skin peptidome of Russian brown frog Rana temporaria.

A nano-HPLC-ESI-OrbiTrap study involving HCD and ETD spectra has been carried out to clarify the composition of the skin peptidome of brown Russian frogs Rana temporaria. This approach allowed determinantion of 76 individual peptides, increasing 3-fold the identified portion of the peptidome in comparison to that obtained earlier with FTICR MS. A search for the new bradykinin related peptides (BRPs) was carried out by reconstructing mass chromatograms based on the ion current of characteristic b- and y-ions. Several peptides were reported in the secretion of R. temporaria for the first time. The overall antibacterial activity of the skin secretion in general and of one individual peptide (Brevinin 1Tb) was determined using PMEU Spectrion (Portable Microbe Enrichment Unit) technology. The inhibitory effects of these peptides on Staphylococcus aureus and Salmonella enterica Serovar typhimutium were equal in scale to that reported for some antibiotics.

Mass spectrometric study of bradykinin-related peptides (BRPs) from the skin secretion of Russian ranid frogs.

Amphibian skin secretion is known to contain biologically active peptides. Bradykinins and related peptides (BRPs) can be found in these animals, while frogs from the genus Rana are considered to be leaders in the levels and variety of these peptides. A reasonable rationalization of this fact is that bradykinins are efficient defense compounds against predators. Forty-four various BRPs have been identified in the skin secretions of five ranid frog species (R. ridibunda, R. lessonae, R. esculenta, R. temporaria, R. arvalis) from the Zvenigorod region (Moscow district, Russia). Some of these peptides are already known, but the novel ones constitute a significant portion. An interesting group of novel peptides was isolated from R. lessonae. These are bradykinin analogues bearing a tyrosine residue in the 5th or 8th position. [Arg(0), Trp(5), Leu(8)]bradykinin and [Thr(6), Leu(8)]bradykinin that had been isolated from fish and avian species, respectively, were also detected in the frog secretion, supporting the predator defense hypothesis. Furthermore, a novel group of BRPs named 'lessonakinins' was discovered in R. lessonae and R. esculenta. All of them include the [Arg(0), Trp(5), Leu(8)]bradykinin sequence and have some structural resemblance to the precursor of this peptide cloned by Chen and coworkers recently. However, the C-terminal part of the lessonakinins does not match the sequence predicted by Chen, demonstrating possible incompleteness of information obtained by cDNA cloning.

Novel natural peptides from Hyla arborea schelkownikowi skin secretion.

Hyla arborea schelkownikowi is one of the leaf frog species inhabiting the southern territories of Russia and the former USSR. This frog species is a member of the Hylidae Rafinesque, 1815 batrachians family. The present study deals with the previously uninvestigated peptidome of the Hyla arborea schelkownikowi skin secretion. Nano-electrospray ionization Fourier transform mass spectrometry (nanoESI-FTMS) of the skin secretion, in the intact form and after acetylation, was selected as the general method of analysis. Electron-capture dissociation (ECD) and collision-induced dissociation (CID) fragmentation were both employed, while de novo sequencing was performed by manual interpretation of the MS data. The suppression of the cyclization of b-ions in the mass spectrometer by the acetylation reaction proved to be very efficient for the de novo sequencing of short peptides. Ten skin peptides were found and all of them, except for bradykinin, had not previously been reported. Six of the peptides belong to the tryptophyllins and related peptides, while three peptides are similar to the aureins.

Mass spectrometric study of peptides secreted by the skin glands of the brown frog Rana arvalis from the Moscow region.

A high-performance liquid chromatography nano-electrospray ionization Fourier transform mass spectrometry (HPLC/nanoESI-FTMS) approach involving recording of collision-activated dissociation (CAD) and electron-capture dissociation (ECD) spectra of an intact sample and two its modifications after performic oxidation and reduction followed by carboxamidomethylation helps to establish peptide profiles in the crude secretion of frog species at mid-throughput level, including de novo sequencing. The proposed derivatization procedures allow increasing of the general sequence coverage in the backbone, providing complementary information and, what is more important, reveal the amino acid sequence in the cystine ring ('rana box'). Thus purely mass spectrometric efficient sequencing becomes possible for longer than usual proteolytic peptides. Seventeen peptides belonging to four known families were identified in the secretion of the European brown frog Rana arvalis inhabiting the Moscow region in Russia. Ranatuerins, considered previously a unique feature of the North American species, as well as a new melittin-related peptide, are worth special mention. The developed approach was previously successfully used for the identification of peptides in the skin secretion of the Caucasian green frog Rana ridibunda.

Electrospray ionization tandem mass spectrometry sequencing of novel skin peptides from Ranid frogs containing disulfide bridges.

Tandem mass spectrometry sequencing, as well as Edman sequencing of peptides belonging to the Rana genus, represents a difficult task due to the presence of a disulfide bridge at the C-terminus and their rather high molecular masses (over 2000 Da). The present study throws light upon the sequence of three rather long peptides (more than 20 amino acid residues each) isolated from the skin secretion of Russian frogs, Rana ridibunda and Rana arvalis. This novel aspect involves the fact that the sequences (including two sequences established de novo) were determined exclusively by means of mass spectrometry. A combination of electron capture dissociation (ECD) and collision-induced dissociaiton (CID) data accompanied by exact mass measurements (LTQ Fourier transform ion cyclotron resonance mass spectrometer) facilitated reaching the goal. To overcome the difficulty dealing with disulphide bridges ("Rana box"), reduction of the S-S bond with dithiotreitol followed by derivatization of Cys residues with iodoacetamide was used. The sequence was determined using combined spectral data on y and b series of fragment ions. A multiple mass spectrometry (MS(3)) experiment was also used to elucidate the sequence inside the "Rana box" after cysteine derivatization. Exact mass measurements were used to differentiate between Lys and Gln residues, while characteristic losses of 29 and 43 Da (d and w fragment ions) in CID and ECD experiments allowed us to distinguish between Ile and Leu isomeric acids.