Hydrophobic Interaction Chromatography Coupled to High-Throughput Venomics for Snake Venom Characterisation with...

Between 80,000 and 140,000 fatalities occur yearly because of snakebite envenoming, which causes it to be classified as a Neglected Tropical Disease (NTD), stressing the importance of snake venom toxin characterisation to aid the development of next-generation antivenoms. Snake venom is made up of a mixture of bioactive proteins that are also known as venom toxins. These venom toxins, being complex and structurally diverse, present a significant challenge for analytical separation and functional characterisation. Traditional methods, such as reversed-phase liquid chromatography (RPLC), often involve denaturing conditions that can alter the native state of these proteins, potentially reducing their bioactivity and limiting subsequent bioassays. Therefore, this study developed and optimised an analytical methodology using non-denaturing Hydrophobic Interaction Chromatography (HIC) for venom separation in conjunction with post-column nanofractionation to perform post-column toxin identification using high-throughput venomics with parallel coagulation bioassays. The results demonstrate that the combination of HIC and nanofractionation allows separation and qualitative profiling of venom toxins with detectable post-column activity, although several toxins co-elute. The post-column bioassays demonstrated detectable coagulation-modulating activity, and high-throughput venomics provided rapid toxin identification capabilities and qualitative information on candidate venom proteins. This approach offers a robust platform for the detailed study of venom toxins, facilitating the discovery of novel bioactive compounds and enhancing our understanding of venom biology.