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Keywords:

  • snake venom;
  • toxins;
  • snake venomics;
  • mass spectrometry;
  • proteomics;
  • cysteine residue;
  • disulfide bond

Abstract

Snake bites can be deadly, but the venoms also contain components of medical and biotechnological value. The proteomic characterization of snake venom proteomes, snake venomics, has thus a number of potential benefits for basic research, clinical diagnosis, and development of new research tools and drugs of potential clinical use. Snake venomics is also relevant for a deep understanding of the evolution and the biological effects of the venoms, and to generate immunization protocols to elicit toxin-specific antibodies with greater specificity and effectiveness than conventional systems. Our snake venomics approach starts with the fractionation of the crude venom by reverse-phase HPLC, followed by the initial characterization of each protein fraction by combination of N-terminal sequencing, SDS-PAGE, and mass spectrometric determination of the molecular masses and the cysteine (SH and S[BOND]S) content. Protein fractions showing a single electrophoretic band, molecular mass, and N-terminal sequence can be straightforwardly assigned by BLAST analysis to a known protein family. On the other hand, protein fractions showing heterogeneous or blocked N-termini are analyzed by SDS-PAGE and the bands of interest subjected to automated reduction, carbamidomethylation, and in-gel tryptic digestion. The resulting tryptic peptides are then analyzed by MALDI-TOF mass fingerprinting followed by amino acid sequence determination of selected doubly and triply charged peptide ions by collision-induced dissociation tandem mass spectrometry. The combined strategy allows us to assign unambiguously all the isolated venom toxins representing over 0.05% of the total venom proteins to known protein families. Protocols and applications of snake venomics are reviewed and discussed. Copyright © 2007 John Wiley & Sons, Ltd.