The venom of cone snails is composed of highly modified peptides (conopeptides) that target a variety of ion channels and receptors. The venom of these marine gastropods represents a largely untapped resource of bioactive compounds of potential pharmaceutical value. Here, we use a combination of bioanalytical techniques to uncover the extent of venom expression variability in Conus purpurascens, a fish-hunting cone snail species. The injected venom of nine specimens of C. purpurascens was separated by reversed-phase high-performance liquid chromatography (RP-HPLC), and fractions were analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) in parallel with liquid chromatography-electrospray ionization (LC-ESI)-TripleTOF-MS to compare standard analytical protocols used in preparative bioassay-guided fractionations with a deeper peptidomic analysis. Here, we show that C. purpurascens exhibits pronounced intraspecific venom variability. RP-HPLC fractionation followed by MALDI-TOF-MS analysis of the injected venom of these nine specimens identified 463 distinct masses, with none common to all specimens. Using LC-ESI-TripleTOF-MS, the injected venom of these nine specimens yielded a total of 5517 unique masses. We also compare the injected venom of two specimens with their corresponding dissected venom. We found 2566 and 1990 unique masses for the dissected venom compared to 941 and 1959 masses in their corresponding injected venom. Of these, 742 and 1004 masses overlapped between the dissected and injected venom, respectively. The results indicate that larger conopeptide libraries can be assessed by studying multiple individuals of a given cone snail species. This expanded library of conopeptides enhances the opportunities for discovery of molecular modulators with direct relevance to human therapeutics. Graphical Abstract The venom of cone snails are extraordinarily complex mixtures of highly modified peptides. Venom analysis requires separation through RP-HPLC followed by MALDI-TOF mass spectrometry or direct analysis using LC-ESI-TripleTOF-MS. Using these techniques, venom intraspecific variability and comparison between injected and dissected were assessed.

α-Conotoxins inhibit nicotinic acetylcholine receptors (nAChRs) and are used as probes to study cholinergic pathways in vertebrates. Model organisms, such as Drosophila melanogaster, express nAChRs in their CNS that are suitable to investigate the neuropharmacology of α-conotoxins in vivo. Here we report the paired nanoinjection of native α-conotoxin PIA and two novel α-conotoxins, PIC and PIC[O7], from the injected venom of Conus purpurascens and electrophysiological recordings of their effects on the giant fiber system (GFS) of D. melanogaster and heterologously expressed nAChRs in Xenopus oocytes. α-PIA caused disruption of the function of giant fiber dorsal longitudinal muscle (GF-DLM) pathway by inhibiting the Dα7 nAChR a homolog to the vertebrate α7 nAChR, whereas PIC and PIC[O7] did not. PIC and PIC[O7] reversibly inhibited ACh-evoked currents mediated by vertebrate rodent (r)α1β1δγ, rα1β1δε and human (h)α3β2, but not hα7 nAChR subtypes expressed in Xenopus oocytes with the following selectivity: rα1β1δε > rα1β1δγ ≈ hα3β2 >> hα7. Our study emphasizes the importance of loop size and α-conotoxin sequence specificity for receptor binding. These studies can be used for the evaluation of the neuropharmacology of novel α-conotoxins that can be utilized as molecular probes for diseases such as, Alzheimer's, Parkinson's, and cancer. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.'

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