Analytical strategies in venomics

Snakebite envenoming is a major issue in certain developing parts of the world, with tens of thousands of deaths and a multitude of lingering permanent medical conditions on a yearly basis as a result. Venoms consist of a plethora of biologically active compounds, mainly peptides and proteins, that can induce these effects. Snake venom characterisation, in terms of identifying and quantifying the venom toxin proteins in crude venoms, can aid in pinpointing the venom toxins responsible for pathologies observed in snakebite victims. It can also assist in developing a new generation of efficient antivenoms. To facilitate this characterisation process, several approaches have been developed, of which venomics, toxicovenomics and antivenomics are the most important ones and aim to map venom toxin composition, their toxic effects, and antivenom efficacy, respectively. Most venomics approaches include peptide-centric analyses of protein digests in combination with advanced separation and detection hardware and involve the use of generic and dedicated software tools for data processing. However, a shift towards protein-level, and even proteoform-level, analysis can be observed over the last few years. A variety of hyphenated intact protein separation techniques, including gel-eluted liquid fraction entrapment electrophoresis, OFFGEL, gel filtration and several (ultra)high pressure and nanoscale chromatographic separations can be named, which allow retention of intact or native states of toxin proteins with ever increasing resolutions. Similarly, rapid detection hardware advances on the mass spectrometry side, and labelling techniques, allow for increasingly sensitive and quantitative venom analysis and characterisation. Most venomics research includes several complementary analytical techniques performed in parallel, with each technique often leading to exclusive protein identifications but also showing significant overlap. The original venomics approach, developed by the Calvete group and termed “Snake venomics”, is used most frequently and is the most profound methodology that has been advanced over the years, thereby resulting in sequentially further identification of venom toxin proteins based on previous results using increasingly in-depth characterisation approaches. The latest advances in this regard nowadays allow for quantitative analysis of all venom toxins in crude venoms, named “Absolute venomics”. This review will focus on the analytical aspects of venomics for which sample preparation, separation, detection and quantitation techniques are discussed. Finally, a comparison between recently published venomics studies is made.