Exploring snake venoms beyond the primary sequence: From proteoforms to protein-protein interactions

Snakebite envenomation has been a long-standing global issue that is difficult to treat, largely owing to the flawed nature of current immunoglobulin-based antivenom therapy and the complexity of snake venoms as sophisticated mixtures of bioactive proteins and peptides. Comprehensive characterisation of venom compositions is essential to better understanding snake venom toxicity and inform effective and rationally designed antivenoms. Additionally, a greater understanding of snake venom composition will likely unearth novel biologically active proteins and peptides that have promising therapeutic or biotechnological applications. While a bottom-up proteomic workflow has been the main approach for cataloguing snake venom compositions at the toxin family level, it is unable to capture snake venom heterogeneity in the form of protein isoforms and higher-order protein interactions that are important in driving venom toxicity but remain underexplored. This review aims to highlight the importance of understanding snake venom heterogeneity beyond the primary sequence, in the form of post-translational modifications that give rise to different proteoforms and the myriad of higher-order protein complexes in snake venoms. We focus on current top-down proteomic workflows to identify snake venom proteoforms and further discuss alternative or novel separation, instrumentation, and data processing strategies that may improve proteoform identification. The current higher-order structural characterisation techniques implemented for snake venom proteins are also discussed; we emphasise the need for complementary and higher resolution structural bioanalytical techniques such as mass spectrometry-based approaches, X-ray crystallography and cryogenic electron microscopy, to elucidate poorly characterised tertiary and quaternary protein structures. We envisage that the expansion of the snake venom characterisation "toolbox" with top-down proteomics and high-resolution protein structure determination techniques will be pivotal in advancing structural understanding of snake venoms towards the development of improved therapeutic and biotechnology applications.