An efficient analytical platform for on-line microfluidic profiling of neuroactive snake venoms towards nicotinic receptor affinity

Abstract

Venomous snakes have evolved their efficient venomous arsenals mainly to immobilize prey. The highly variable toxic peptides in these venoms target a myriad of neurotoxic and haemotoxic receptors and enzymes and comprise highly interesting candidates for drug discovery. Discovery of bioactive compounds from snake venoms, however, is a challenge to achieve. We have developed and applied a methodology to rapidly assess bioactives in a snake venom proteome. Our microfluidic platform opens up efficient and rapid profiling of venomous anti-cholinergic receptor compounds. The key advantages of our methodology are: (i) nano amounts of venom needed; and (ii) a direct correlation of selected bioaffinities with accurate mass. To achieve this, we have for the first time successfully constructed a functional post nano-LC split to MS and bioaffinity profiling. In our method, comprehensive venom profiles with accurate masses and corresponding bioaffinities are obtained in one analytical run and will subsequently allow immediate purification of bioactive peptides with LC–MS, guided by accurate masses of the bioactives only. We profiled several neurotoxic Elapidae snake venoms using our methodology in combination with the acetylcholine binding protein (AChBP) as biological target protein. The latter is a homologue of nicotinic acetylcholine receptors (nAChRs), a drug target in neurodegenerative diseases and cognitive decline such as Parkinson's and Alzheimer's, and in pain related diseases. Our methodology was evaluated and validated with high-affinity α-bungarotoxin and haemotoxic/proteolytic Vipera ammodytes venom spiked with α-bungarotoxin. Thereafter, the methodology was applied to profile the venom proteomes of Dendroaspis jamesoni kaimosae, Naja annulifera and Naja nivea. Gathering comprehensive profiling data took less than 2 h per snake venom measured. The data yielded 20 AChBP ligands of which the corresponding accurate masses were used to retrieve information from literature regarding their function and targeting specificity. We found that from these 20 ligands, 11 were previously reported on, while information on the others could not be found. From these 11 peptides, five have been reported to have nAChR affinity, while the others are reported as cytotoxic, cardiotoxic or as orphan toxin. Our methodology has the potential to aid the field of profiling complex animal venoms for drug discovery.