Snake venoms are composed of bioactive proteins and peptides from various toxin families and elicit potent pharmacological activity. There is great interest in characterising these venom proteins for the development of effective antivenom treatment as well as utilisation for biomedical and therapeutic applications. However, a thorough structural understanding of the snake venom proteins is necessary. Higher-order protein complexes are known to form in snake venoms and lend structural and functional diversity, often eliciting greater activity than the sum of monomeric protein species. Despite the significance, the nature of these protein complexes is extremely underexplored. In this study, we demonstrate the use of mass spectrometry (MS)-based strategies to explore the toxins at a quaternary level in the venom from the medically significant forest cobra (Naja melanoleuca). Small toxins, mainly three finger toxins (3FTxs) and phospholipase A2s (PLA2s), were identified by comparison of intact and chemically reduced masses using matrix-assisted laser desorption ionisation (MALDI-MS) profiling. Notably, interrogation of these small toxins by native MS and collision-induced dissociation revealed the presence of various non-covalent 3FTx and PLA2 dimers, providing insight on the higher-order protein structures for a variety of N. melanoleuca toxins using a MS-based approach. Furthermore, phospholipid substrate specificity of N. melanoleuca PLA2 enzymes were explored, capturing the indiscriminate activity of these PLA2s towards a range of phospholipid classes for the first time.
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