Intraspecific variations in Conus purpurascens injected venom using LC/MALDI-TOF-MS and LC-ESI-TripleTOF-MS

Abstract

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.

Conotoxin Diversity in Chelyconus ermineus (Born, 1778) and the Convergent Origin of Piscivory in the Atlantic and Indo-Pacific Cones

“…Therefore, it is very likely that even the number here reported underestimates the whole diversity of conotoxins produced by this species. Such interindividual differences are congruent with results reported in the closely related C. purpurascens ( Rodriguez et al. 2015 ) and for three specimens of D. betulinus with different body sizes ( Peng et al.…”

Accelerated proteomic visualization of individual predatory venoms of Conus purpurascens reveals separately evolved predation-evoked venom cabals

“…The ability of cone snails to switch between separately evolved predatory and defensive venom regimes appears to underpin this remarkable structural and functional diversity. However, it remains unclear what contributes to their remarkable inter-and intra-species variability 6 , 10 – 13 .…”

“…Given the surprising individual variability observed in C. purpurascens injected predatory venom 11 , 13 , we were interested in determining if the cabals identified previously in their pooled predatory venom were present in individual C. purpurascens . In this study, injected predatory venom from nine C. purpurascens was analyzed by LC-ESI-triple-TOF-MS to determine the prey capture strategy used by each specimen.…”

Ecological venomics: How genomics, transcriptomics and proteomics can shed new light on the ecology and evolution of venom

“…Limited Top-Down quantitation of MS1 spectra of venoms has been published previously using label free quantitation [16,123].…”