PROTEOMIC APPROACH APPLIED TO THE STUDY OF PEPTIDES FROM THE BRAZILIAN TREE-FROG Hyla biobeba SKIN SECRETION

Fontes, W.(2), Matsushita, R.H.(1), Sousa, M.V.(2), Schwartz, C.A.(1), Schwartz, E.F.(1), Sebben, A.(1), Castro, M.S.(1,2)

(1)Laboratório de Toxinologia, Departamento de Ciências Fisiológicas, IB, Universidade de Brasília; (2)Laboratório de Bioquímica e Química de Proteínas, Centro Brasileiro de Serviços e Pesquisas em Proteínas, Departamento de Biologia Celular, IB, Universidade de Brasília, Brasília, DF, Brasil.

Amphibian skin secretions have been considered a rich source of bioactive compounds, such as biogenic amines, steroids, alkaloids, peptides and proteins. Biologically active peptides represent a significant part of this repertoire of substances and they act on different physiological processes, including passive defense against microorganisms. In order to evaluate the diversity of peptides present in amphibian skin secretions, we applied a proteomic approach to improve the number of peptides identified from the skin secretion of the treefrog Hyla biobeba, commonly found in our region. Adult specimens of Hyla biobeba were collected from Distrito Federal region and their skin secretions were obtained by mild electrical stimulation, dissolved in water and lyophilized. Dried secretion aliquots were applied onto a C₁₈ reversed-phase column (Vydac 218TP54, 4.6 x 250 mm, Separations Group, USA) and fractionated using a linear gradient of acetonitrile. Eluted fractions were manually collected, vacuum-dried and submitted to mass spectrometric analysis using a MALDI-TOF spectrometer (Reflex IV, Bruker). The samples were prepared using acyano-4-hydroxy-cinnamic acid (20 mg/mL) as matrix dissolved in acetonitrile:0.1% TFA (1:2) and applied onto the target by dry-droplet method. The m/z used range was 0-3 kDa. This kind of analysis permitted the rapid homogeneity assessment of most chromatographic fractions and also the determination of their molecular masses. Some of these peptides were selected for further purification and structural characterization.

CORRESPONDENCE TO:

Fontes, W., Laboratório de Bioquímica e Química de Proteínas, Centro Brasileiro de Serviços e Pesquisas em Proteínas, Departamento de Biologia Celular, IB, Universidade de Brasília, Brasília, 70910-900, DF, Brasil. Email: wagnerf@unb.br