Analysis of the salivary gland proteins from leech Haementeria depressa using bidimensional electrophoresis and MALDI-TOF-MS methodologies

Ricci-Silva, M.E.(1), Stocklin, R.(2), Rádis-Baptista, G.(3), Yamane, T.(3), Favreau, P.(2), Michalet, S.(3), Chudzinski-Tavassi, A.M.(1)

(1)Departamento de Bioquímica, Instituto Butantan, São Paulo, Brasil, (2)Atheris Laboratory, Genebra, Suíça, (3)Toxinologia Molecular, Instituto Butantan, São Paulo; Brasil.

The salivary extract of the Haementeria depressa leech contains several important proteins acting on blood coagulation and on the fibrino(geno) lytic systems. The two major proteins described so far from this species include the fibrinogenolytic metalloproteinase hementerin and the factor Xa inhibitor lefaxin. Our aim is to further investigate the protein and peptide content of this extract using Proteomics and Mass Spectrometry (MS) strategies. The bidimensional electrophoresis technique showed 96 spots above 25 kDa, from 133 total spots in the extract 1, indicating the feasibility of this methodology for the identification of large proteins. Preliminary study based on MALDI-TOF-MS will be also presented here. The optimization of a suitable matrix-solvent-extract combination was followed by appropriate adjustment of the instruments’ settings on a VoyagerÒ STR Biospectrometry (Applied Biosystem) MALDI-TOF mass spectrometer providing a good compromise between sensitivity and resolution. The sample was analysed using different matrices in linear mode with delayed extraction, leading to detection of several compounds with molecular masses of interest. The main monocharged ions of m/z detected were: 2028.37, 3369.83, 3864.95, 4687.60, 5103.36, 10805.65, 14046.26, 17165.44, 28261.10, 29261.10, 29068.21 and 42318.58. Isolation, purification and further structural investigations of these compounds are presently under investigation with the aim of discovering novel bioactive compounds.

Supported by FAPESP. 

CORRESPONDENCE TO:

MARIA ESTHER RICCI DA SILVA, Avenida Vital Brasil 1500, Butantan, São Paulo, SP, CEP: 05503900, Brasil, Email: esthericci@hotmail.com