T. Yu. Samgina

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 (MS3) 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.

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 spectral study of the skin peptide of brown frog Rana temporaria 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.

Investigation of skin secretory peptidome of Rana lessonae frog by mass spectrometry

Amphibian skin secretion represents a cerain scientific interest as a source of biologically active natural peptides. In the present research skin peptidome of wide-spread European frog Rana lessonae (Camerano, 1882) was studied for the first time ever. Peptide sequencing was accomplished with Fourier transform ion cyclotron resonance mass spectrometer in collision-induced and electron capture dissociation modes. A portion of amphibian peptides contains intramolecular C-terminal disulfide cycle which obstructs mass spectrometric sequencing. Two methods were utilized to overcome this difficulty: reduction with dithiotreithol followed by thiol group alkylation and oxidation into sulfonic acid groups with performic acid. Integrated approach employed in the present study allowed the identification of 49 peptides (of 6 to 37 amino acid residues), including 19 novel species.

New cysteine-modifying reagents: Efficiency of derivatization and influence on the signals of the protonated molecules of disulfide-containing peptides in matrix-assisted laser desorption/ionization mass spectrometry

Mass spectrometric de novo sequencing of skin secretion peptides from genus Rana is complicated because of C-terminal disulfide cycles present in their structure. Brevinin-1E and brevinin-2Ec from the skin secretion of the Marsh Frog R. ridibunda were used for a comparative study of six N-phenylmaleimide derivatives as new alkylating agents for cysteine thiol moieties. The paper describes the synthesis and confirmation of the structures of the obtained compounds. A procedure was developed for modifying thiol groups with the proposed reagents. Alkylation efficiency and the effect on the peak intensity in matrix-assisted laser desorption/ionization (MALDI) spectra were investigated. The best results were obtained for 2,4- and 2,5-dimethylphenylmaleimides. Additionally tested iodoacetic acid was shown to be a powerful modifier of thiol groups, while its excess notably increases the intensities of the peaks of protonated molecules in the MALDI mass spectra of both peptides.

HPLC and MALDI investigation of the stress influence on the composition of skin secretion of the Common frog Rana temporaria

Skin secretory amphibian antimicrobial peptides are the part of their immune defense. The present work is devoted to the study of the influence of “water environment stress” and additional bacterial impact on the composition of the skin secretion of the Common frog (Rana temporaria) by means of high-performance liquid chromatography (HPLC) and matrix assisted laser desorption/ionization (MALDI) mass spectrometry. It was shown that the contact of the amphibian species with Micrococcus luteus and Staphylococcus aureus stimulates the release of antimicrobial peptides, maintains the high bradykinin and related peptides levels in the skin secretion and influences the processing of the latter ones. The possibilities of mass spectrometric profiling by using HPLC and MALDI were demonstrated. This feature allows the detection of potentially bioactive peptides for their future direct testing, as has been shown for temporin M and brevinin 1Tb in the present study.

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.

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 Β 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 α esters. The principal characteristic fragment ions by means of which such compounds in liquid-crystal mixtures can be identified and quantitatively determined were established.

Mass spectra of arylalkylpyridines

The basic principles of the mass-spectral fragmentation of arylalkylpyridines that contain substituents in various positions were established. The principle ions that can be used in Chromatographic mass-fragmentographic analysis in the study of liquid-crystal compositions were determined for each specific case.