The proteome composition of snake venom alters with age and therefore the developmental stage of the organism, which donated the specimen, should be taken into account. To this end, we performed a comparative analysis of B. atrox venom proteome in three stages of maturity. To our knowledge, this is the first proteome analysis of snake venoms associated with the ontogenetic development, although other proteomic studies on snake venom have been published previously [13,14].
In a typical proteomic routine, proteins are separated by 2-DE, visualized by silver or Coomassie blue staining  and protein spots in-gel digested with trypsin . An aliquot of the digest is subjected to PMF by MALDI-TOF mass spectrometry. If no conclusive identification is achieved, tryptic peptides are then extracted from the gel matrix and sequenced by tandem mass spectrometry . However, in both protein identification approaches it is required that the exact sequence of the analyzed protein is available in a database. Nevertheless, only a small number of B. atrox proteins are currently available in a sequence database and therefore the scope of protein identification was very limited. To address this issue, we complemented conventional protein identification methods, which are based on the exact matching of tandem mass spectra to database sequences, with sequence-similarity searches. The latter approach enabled confident identification of unknown proteins that only distantly related to known proteins from other species [18-20]. Using a combination of stringent and sequence-similarity database searches, we identified all major components of venoms and mapped out changes in the abundance of individual protein components in ontogenetically altered proteomes.
The natural polymorphism of the protein sequences, together with the absence of a complete and annotated snake genome, which could be used as a referenceid not allow the unambiguous assignment of the most related protein homologues due to the low sequence coverage. However, the group-specific assignment, based on the peptide sequences, was always reliable.
Based on peptide mass fingerprinting analysis we classified the spots of the 2-DE maps in 27 groups. Most groups and those with larger number of spots corresponded to proteinases (metallo and serine proteinases). This result agrees with the fact that venoms from Bothrops species are haemotoxic and promote haemorrhaging primary to extensive local swelling and necrosis .
The proteome maps also enabled the identification of new groups of potential ontogenetic molecular markers. For instance, we found that some groups of proteins including P-III class metalloproteinases (L, M, O and P), serine proteinases (N, Q and R) are more abundant in juvenile specimens, while metalloproteinases from class P-I (group D) are more expressed in adults.
Overall, more groups of proteins were identified in the proteome maps from juveniles, than in adults. Proteome maps from sub-adults contained spots from both juveniles and adults, along with a few stage-specific spots. Previous work suggested that the B. atrox venoms from young individuals trigger more potent biological effects than adult venoms [2,5]. The decrease in hemorrhagic activity in B. atrox venoms during ontogenetic development may be explained by the lower levels of Zn-metalloproteinases of P-III class in adults comparing to juveniles. On the other hand, the higher concentration of berythractivase, a pro-thrombin activator, may reflect the higher coagulant activity of juvenile venoms. The effect of VEGFs, NGFs and CRISPs, all more expressed in young specimens, certainly also contribute to the higher pharmacological activities of the juvenile venom.
The diversity in the protein composition and biological activity of snake venom during growth may be related to adaptation by evolutionary processes to the type and size of the prey [21-24]. Young Bothrops snakes preferentially eat amphibians, lizards, birds, and shift to mammals when they become adults . Therefore, the qualitative and quantitative changes in the B. atrox venom proteome are most likely related to the survival of the snake by prey adaptation.