Animal venoms contain a significant contribution of proteins and peptides. Co-evolution of venomous animals has resulted in a diversity of biologically active venom proteins, which are integral to an animal’s defence and in subduing prey. Snake venoms more specifically contain a variety of bioactive proteins and peptides possessing pharmacological and toxicological activities; most dominantly in the form of phospholipases A2, metalloproteinases, serine proteases and three-finger peptides. Due to the composition, exposure to venom by snake bite can induce hemotoxic, neurotoxic and cytotoxic effects in the bitten animal. The successful diagnosis of snake bite, including the definitive determination of species of origin, and thus successful path to treatment, can be erroneous. Forensic and clinical toxicology laboratories have historically relied upon immunoassay to determine the presence or absence of snake venom.
Proteomics techniques, employing high resolution mass spectrometry (MS) can be used to achieve selectivity and specificity for the detection of target venom proteins, therefore enabling the unequivocal confirmation of snake bites, and the originating species possible. Here we report the development of a novel methodology using bottom-up proteomics, for species identification from venom proteins. Preparation of venom proteins by reduction/alkylation and tryptic digestion was achieved before analysis by liquid chromatography mass spectrometry (LC-MS) using a Thermo Fisher QE Plus Orbitrap MS. Data were acquired using the TraceFinderTM software and Parallel Reaction Monitoring (PRM), with protein identification achieved using a Serpentes fasta subset of the UniProtKB database, processed through Proteome Discover software (version 2.2).
The established methodology permits the definitive confirmation of a snake bite, uniquely detecting and identifying nine snake venoms using LC-MS. This work reports the first case of the unequivocal identification of the Eastern Brown snake venom in a swab taken from a person who had succumbed to the snake bite. The method provides an advancement in forensic coronial toxicology and highlights the potential of proteomics assays to routine forensics investigations.