title: A ray of venom: Combined proteomic and transcriptomic investigation of fish venom composition using barb tissue from the blue-spotted stingray (Neotrygon kuhlii).
abstract: Fish venoms remain almost completely unstudied despite the large number of species. In part this is due to the inherent nature of fish venoms, in that they are highly sensitive to heat, pH, lyophilisation, storage and repeated freeze-thawing. They are also heavily contaminated with mucus, which makes proteomic study difficult. Here we describe a novel protein-handling protocol to remove mucus contamination, utilising ammonium sulphate and acetone precipitation. We validated this approach using barb venom gland tissue protein extract from the blue-spotted stingray Neotrygon kuhlii. We analysed the protein extract using 1D and 2D gels with LC-MS/MS sequencing. Protein annotation was underpinned by a venom gland transcriptome. The composition of our N. kuhlii venom sample revealed a variety of protein types that are completely novel to animal venom systems. Notably, none of the detected proteins exhibited similarity to the few toxin components previously characterised from fish venoms, including those found in other stingrays. Putative venom toxins identified here included cystatin, peroxiredoxin and galectin. Our study represents the first combined survey of gene and protein composition from the venom apparatus of any fish and our novel protein handling method will aid the future characterisation of toxins from other unstudied venomous fish lineages.
  
  
  CONCLUSIONS
  These results show an efficient manner for removing mucus from fish venoms. These results are the first insights into the evolution of proteins present on stingrayvenom barbs.
authors.affiliation: Venom Evolution Laboratory, School of Biological Sciences, University of Queensland, St Lucia, Qld 4072, Australia.
authors.forename: Kate
authors.initials: K
authors.lastname: Baumann
authors.affiliation: Venom Evolution Laboratory, School of Biological Sciences, University of Queensland, St Lucia, Qld 4072, Australia; Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool L3 5QA, UK; Molecular Ecology and Evolution Group, School of Biological Sciences, Bangor University, Bangor LL57 2UW, UK.
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Casewell
authors.affiliation: Venom Evolution Laboratory, School of Biological Sciences, University of Queensland, St Lucia, Qld 4072, Australia; HRJ Research Institute of Chemistry, International Centre for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi 75270, Pakistan.
authors.forename: Syed A
authors.initials: SA
authors.lastname: Ali
authors.affiliation: Venom Evolution Laboratory, School of Biological Sciences, University of Queensland, St Lucia, Qld 4072, Australia; Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: Timothy N W
authors.initials: TN
authors.lastname: Jackson
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072, Australia; School of Pharmacy, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: Irina
authors.initials: I
authors.lastname: Vetter
authors.affiliation: Venom Evolution Laboratory, School of Biological Sciences, University of Queensland, St Lucia, Qld 4072, Australia.
authors.forename: James S
authors.initials: JS
authors.lastname: Dobson
authors.affiliation: Venom Evolution Laboratory, School of Biological Sciences, University of Queensland, St Lucia, Qld 4072, Australia.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Cutmore
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, Qld 4072, Australia.
authors.forename: Amanda
authors.initials: A
authors.lastname: Nouwens
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: Vincent
authors.initials: V
authors.lastname: Lavergne
authors.affiliation: Venom Evolution Laboratory, School of Biological Sciences, University of Queensland, St Lucia, Qld 4072, Australia; School of Pharmacy, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: Bryan G
authors.initials: BG
authors.lastname: Fry
mesh_terms: D000818:Animals; D029941:Fish Proteins / Q000096:biosynthesis*; D005397:Fish Venoms / Q000096:biosynthesis*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D040901:Proteomics; D012860:Skates, Fish / Q000378:metabolism*
chemical_list: D029941:Fish Proteins; D005397:Fish Venoms
keywords: 1D gel; 2D gel; Proteome; Transcriptome; Venom