17th International Mass Spectrometry Conference :: Prague, 2006
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|Presentation date:||Thu, Aug 31, 2006|
|Presentation time:||14:30 – 16:00|
Tatiana Yurievna Samgina1, Konstantin Alexandrovich Artemenko1, Roman Alexandrovich Zubarev2, Albert Tarasovich Lebedev11 Moscow State University, Moscow, Russian Federation
Correspondence address: Tatiana Yurievna Samgina, Moscow State University, Organic Chemistry, Leninskie Gory 1/3, Moscow, 119992 Russian Federation.
Keywords: Digests, Enzymatic or Proteolytic; MS/MS, Structure Determination; Peptide Structure; Proteomic.
Novel aspect: MALDI analysis of crude peptide secretion was optomised. Variations of peptide profile of the same Rana species were observed.
The secretion of amphibian granular skin glands is the known source of bioactive compounds which are the first defensive line for animal against pathogenic microorganisms and predators. Peptides are the main portion of crude secretions of Rana species. Some of them possess pronounced antimicrobial activity towards gram-positive and gram-negative bacteria. Besides they show antifungal activity. Every species of Rana family has its unique peptide profile. Moreover peptide profile may be different for the same species leaving far away from one another (thousands kilometers) due to various leaving conditions. The aim of the present study deals with the comparative study of peptide profiles of Marsh frogs Rana ridibunda from Moscow region and The North Caucasus. HPLC, MALDI TOF and LC-ESI-MS-MS were used for this purpose.
Female Rana ridibunda species were caught in Moscow region and Abkhazia (North Caucasus). Crude secretions were obtained by the electrostimulations of dorsal skin glands. Further separation and purification were performed with charge reversed HPLC. Molecular masses of the peptides were recorded with MALDI TOF AutoFlex instrument. MS/MS experiments were done with Thermo Finnigan LTQ FT instrument. To destroy disulphide bridge and get derivatives the corresponding peptides were consecutively reacted with dithiothreitol and iodoacetamide.
Earlier we have demonstrated that the main peptides of Rana ridibunda species from Moscow region are antimicrobial peptides from brevinin-1E, brevinin-2E, esculentin-1 families with 7-member C-terminal ring. Among them are as the known peptides (brevinin-1E, brevinin-2Ec, esculentin-1a) and the new ones (with molecular masses 2636,37; 2934,55; 2663,49, 3824,07Da). Both frogs (Caucasian and Moscow) possess similar peptide profile, i.e.all the peptides identified in the skin secret of the Moscow frog are present in the skin secret of the Abkhazian frog living 1500 km to the south. The main difference deals with the ratio of the main components. Thus brevinin-1E, which is the dominant component, constitutes 50% of the peptidesí weight in the case of Moscow grog and only 25% in the case of the Abkhazian one. On the other hand the major component of the crude secretion of the frog from Abkhazia is brevenine-2Ec (40-45% of the total amount), constitutes only 25% in the case of the Northern species. The amount of other principal peptides is about the same for the both species. Nevertheless a number of unique minor peptides were identified in each of the studied species. These peptides may be characteristic for the living conditions of the frogs.
In parallel with the principal study the conditions of recording of MALDI spectra of crude secretions (without preliminary separation) were optimized.