Exploring Top-Down\nMass Spectrometric Approaches To\nProbe Forest Cobra (<i>Naja melanoleuca</i>) Venom Proteoforms

Snake\nvenoms are comprised of bioactive proteins and peptides that\nfacilitate severe snakebite envenomation symptoms. A comprehensive\nunderstanding of venom compositions and the subtle heterogeneity therein\nis important. While bottom-up proteomics has been the well-established\napproach to catalogue venom compositions, top-down proteomics has\nemerged as a complementary strategy to characterize venom heterogeneity\nat the intact protein level. However, top-down proteomics has not\nbeen as widely implemented in the snake venom field as bottom-up proteomics,\nwith various emerging top-down methods yet to be developed for venom\nsystems. Here, we have explored three main top-down mass spectrometry\nmethodologies in a proof-of-concept study to characterize selected\nthree-finger toxin and phospholipase A<sub>2</sub> proteoforms from\nthe forest cobra (Naja melanoleuca)\nvenom. We demonstrated the utility of a data-independent acquisition\nmode “MS<sup>E</sup>” for untargeted fragmentation on\na chromatographic time scale and its improvement in protein sequence\ncoverage compared to conventional targeted tandem mass spectrometry\nanalysis. We also showed that protein identification can be further\nimproved using a hybrid fragmentation approach, combining electron-capture\ndissociation and collision-induced dissociation. Lastly, we reported\nthe promising application of multifunctional cyclic ion mobility separation\nand post-ion mobility fragmentation on snake venom proteins for the\nfirst time.