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. Copyright © 2009 John Wiley & Sons, Ltd.     

Bioactive peptides from the skin of ranid frogs: modern approaches to the mass spectrometric <Emphasis Type="Italic">de novo</Emphasis> sequencing

A mass spectrometric approach to the de novo sequencing of peptides containing up to 50 amino acid residues was proposed. Arising problems of the mass spectrometric sequencing and their solution were considered on specific examples. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1061–1072, May, 2008.     

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.     

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.     

Mass spectral study of the skin peptide of brown frog <Emphasis Type="Italic">Rana temporaria</Emphasis> from Zvenigorod population

Skin secretions of amphibian are an interesting source of biologically active peptides. The present study provides the profile of the skin secretions of the brown frog Rana temporaria from Zvenigorod population (Russia). Sequencing of the skin secretion components has been carried out on an ion cyclotron resonance instrument with electrospray ionization and two methods of fragmentation activation, collisional activation and electron capture. For sequencing of the peptides containing intermolecular C-terminal disulfide cycle two methods of disulfide bond opening have been used: reduction with subsequent alkylation of the free thiol groups and oxidation with performic acid with the formation of sulfo-acid groups. The peptide profile of Rana temporaria studied by a complex mass spectral method has been compared with the data for the frogs of other European populations of this species. For the first time we have revealed ornithokinin-antagonist of the ornithokinin receptor-in skin secretions of amphibians.     

Mass spectra of 4,5-disubstituted 1,2,3-thiadiazoles

The schemes of the fragmentation of 4-substituted 5-arylamino-1,2,3-thiadiazoles were established on the basis of electron-impact mass spectra and high-resolution mass-spectrometric data, and the effect of substituents on the fragmentation pathways was analyzed. It is shown that the cyclization of the [M-N₂]⁺ ions formed in the fragmentation of compounds that contain a thiocarboxamido group in the 4 position proceeds with the participation of the most nucleophilic heteroatom.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 1, pp. 118–122, January, 1988.     

Mass spectra of liquid crystals. 2. Esters of alkoxy-substituted nicotinic and picolinic acids

Esters of alkoxypicolinic and alkoxynicotinic acids are reliably identified by means of their mass spectra and PMR spectra. While the alkoxypyridoyl cation in the case of the esters undergoes fragmentation only with the successive splitting out of an olefin via the McLafferty mechanism and then a CO molecule, these processes also take place in the reverse order in the case of the esters. The principal characteristic fragment ions by means of which such compounds in liquid-crystal mixtures can be identified and quantitatively determined were established.See [1] for Communication 1.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 5, pp. 637–641, May, 1988.