Bothrops atrox is responsible for the majority of snake bite accidents in the Brazilian Amazon region. Previous studies have demonstrated that the biological and pharmacological activities of B. atrox venom alter with the age of the animal. Here, we present a comparative proteome analysis of B. atrox venom collected from specimens of three different stages of maturation, juveniles, sub-adults and adults. The comparative analysis identified remarkable differences in protein expression of several genes that correlate well with previously reported alterations in the venom activity upon ontogenetic development. Optimized conditions for two-dimensional gel electrophoresis (2-DE) of pooled venom samples were achieved using immobilized pH gradient (IPG) gels of non-linear 3-10 pH range during the isoelectric focusing step and 10-20% gradient polyacrylamide gels in the second dimension. Computational image analysis of the 2-DE gels demonstrated differences in spot number and intensity among juvenile, sub-adult and adult samples. Although peptide mass fingerprinting (PMF) failed to identify even a minor fraction of spots it, allowed us to group spots that displayed similar peptide maps. The spots were subjected to a combination of tandem mass spectrometry and Mascot and MS BLAST database searches that identified several classes of proteins, including metalloproteinases, serine proteinases, lectins, phospholipases A₂, L-amino oxidases, nerve growth factors, vascular endothelial growth factors and cysteine-rich secretory proteins.

The analysis B. atrox samples from specimens of different ages by 2-DE confirmed that venom proteome alters upon ontogenetic development. We identified stage specific and differentially expressed polypeptides that may be responsible for the activities of the venom in each developmental stage. The results provide insight into the molecular basis of the relation between symptomatology of snake bite accidents in humans and the venom composition.