Interrogating the higher order structures of snake venom proteins using an integrated mass spectrometric approach

Snake venoms contain complex mixtures of proteins vital for the survival of venomous snakes. Aligned with their diverse pharmacological activities, the protein compositions of snake venoms are highly variable, and efforts to characterise the primary structures of such proteins are ongoing. Additionally, a significant knowledge gap exists in terms of the higher-order protein structures which modulate venom potency, posing a challenge for successful therapeutic applications. Here we use a multifaceted mass spectrometry approach to characterise proteins from venoms of Collett's snake Pseudechis colletti and the puff adder Bitis arietans. Following chromatographic fractionation and bottom-up proteomics analysis, native mass spectrometry identified, among other components, a non-covalent L-amino acid oxidase dimer in the P. colletti venom and a C-type lectin tetramer in the B. arietans venom. Furthermore, a covalently-linked phospholipase A(2) (PLA(2)) dimer was identified in P. colletti venom, from which the PLA(2) species were shown to adopt compact geometries using ion mobility measurements. Interestingly, we show that the dimeric PLA(2) possesses greater bioactivity than the monomeric PLA(2)s. This work contributes to ongoing efforts cataloguing components of snake venoms, and notably, emphasises the importance of understanding higher-order venom protein interactions and the utility of a combined mass spectrometric approach for this task. Significance: The protein constituents of snake venoms represent a sophisticated cocktail of biologically active molecules ideally suited for further exploration in drug design and development. Despite ongoing efforts to characterise the diverse protein components of such venoms there is still much work required in this area, particularly in moving from simply describing the protein primary sequence to providing an understanding of quaternary structure. The combined proteomic and native mass spectrometry workflow utilised here gives new insights into higher order protein structures in selected snake venoms, and can underpin further investigation into the protein interactions which govern snake venom specificity and potency.