title: An in-depth snake venom proteopeptidome characterization: Benchmarking Bothrops jararaca.
abstract: A large-scale proteomic approach was devised to advance the understanding of venom composition. Bothrops jararaca venom was fractionated by OFFGEL followed by chromatography, generating peptidic and proteic fractions. The latter was submitted to trypsin digestion. Both fractions were separately analyzed by reversed-phase nanochromatography coupled to high resolution mass spectrometry. This strategy allowed deeper and joint characterizations of the peptidome and proteome (proteopeptidome) of this venom. Our results lead to the identification of 46 protein classes (with several uniquely assigned proteins per class) comprising eight high-abundance bona fide venom components, and 38 additional classes in smaller quantities. This last category included previously described B. jararaca venom proteins, common Elapidae venom constituents (cobra venom factor and three-finger toxin), and proteins typically encountered in lysosomes, cellular membranes and blood plasma. Furthermore, this report is the most complete snake venom peptidome described so far, both in number of peptides and in variety of unique proteins that could have originated them. It is hypothesized that such diversity could enclose cryptides, whose bioactivities would contribute to envenomation in yet undetermined ways. Finally, we propose that the broad range screening of B. jararaca peptidome will facilitate the discovery of bioactive molecules, eventually leading to valuable therapeutical agents.
  
  
  
  Our proteopeptidomic strategy yielded unprecedented insights into the remarkable diversity of B. jararaca venom composition, both at the peptide and protein levels. These results bring a substantial contribution to the actual pursuit of large-scale protein-level assignment in snake venomics. The detection of typical elapidic venom components, in a Viperidae venom, reinforces our view that the use of this approach (hand-in-hand with transcriptomic and genomic data) for venom proteomic analysis, at the specimen-level, can greatly contribute for venom toxin evolution studies. Furthermore, data were generated in support of a previous hypothesis that venom gland secretory vesicles are specialized forms of lysosomes. Two testable hypotheses also emerge from the results of this work. The first is that a nucleobindin-2-derived protein could lead to prey disorientation during envenomation, aiding in its capture by the snake. The other being that the venom's peptidome might contain a population of cryptides, whose biological activities could lead to the development of new therapeutical agents.
authors.affiliation: Laboratory of Toxinology, Oswaldo Cruz Institute, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil; National Institute of Science and Technology on Toxins (INCTTOX), CNPq, Brazil.
authors.forename: Carolina A
authors.initials: CA
authors.lastname: Nicolau
authors.affiliation: Laboratory of Toxinology, Oswaldo Cruz Institute, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil; Laboratory for Proteomics and Protein Engineering, Carlos Chagas Institute, Fundação Oswaldo Cruz, PR, Brazil.
authors.forename: Paulo C
authors.initials: PC
authors.lastname: Carvalho
authors.affiliation: Laboratório Especial de Toxinologia Aplicada, Instituto Butantan, SP, Brazil.
authors.forename: Inácio L M
authors.initials: IL
authors.lastname: Junqueira-de-Azevedo
authors.affiliation: Laboratory of Toxinology, Oswaldo Cruz Institute, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil; National Institute of Science and Technology on Toxins (INCTTOX), CNPq, Brazil.
authors.forename: André
authors.initials: A
authors.lastname: Teixeira-Ferreira
authors.affiliation: Proteomics Unit, Rio de Janeiro Proteomics Network, Departamento de Bioquímica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; National Institute of Science and Technology on Toxins (INCTTOX), CNPq, Brazil.
authors.forename: Magno
authors.initials: M
authors.lastname: Junqueira
authors.affiliation: Laboratory of Toxinology, Oswaldo Cruz Institute, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil; National Institute of Science and Technology on Toxins (INCTTOX), CNPq, Brazil.
authors.forename: Jonas
authors.initials: J
authors.lastname: Perales
authors.affiliation: Laboratory of Toxinology, Oswaldo Cruz Institute, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil; National Institute of Science and Technology on Toxins (INCTTOX), CNPq, Brazil.
authors.forename: Ana Gisele C
authors.initials: AG
authors.lastname: Neves-Ferreira
authors.affiliation: Laboratory of Toxinology, Oswaldo Cruz Institute, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil; National Institute of Science and Technology on Toxins (INCTTOX), CNPq, Brazil. Electronic address: richardhemmi@gmail.com.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Valente
mesh_terms: D000818:Animals; D019985:Benchmarking; D017837:Bothrops; D002135:Calcium-Binding Proteins / Q000032:analysis; D003435:Crotalid Venoms / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000032:analysis; D008247:Lysosomes / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000032:analysis; D000080866:Nucleobindins; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000032:analysis; D020543:Proteome / Q000032:analysis*
chemical_list: D002135:Calcium-Binding Proteins; D003435:Crotalid Venoms; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D010455:Peptides; D011506:Proteins; D020543:Proteome
keywords: Bothrops jararaca; Cryptome; OFFGEL; Proteomics; Proteopeptidome; Snake venomics