Proteomic Analysis Provides Insights on Venom Processing in Conus textile

Abstract

Conus species of marine snails deliver a potent collection of toxins from the venom duct via a long proboscis attached to a harpoon tooth. Conotoxins are known to possess powerful neurological effects and some have been developed for therapeutic uses. Using mass-spectrometry based proteomics, qualitative and quantitative differences in conotoxin components were found in the proximal, central and distal sections of the C. textile venom duct suggesting specialization of duct sections for biosynthesis of particular conotoxins. Reversed phase HPLC followed by Orbitrap mass spectrometry and data analysis using SEQUEST and ProLuCID identified 31 conotoxin sequences and 25 post-translational modification (PTM) variants with King-Kong 2 peptide being the most abundant. Several previously unreported variants of known conopeptides and were found and this is the first time that HyVal is reported for a disulfide rich Conus peptide. Differential expression along the venom duct, production of PTM variants, alternative proteolytic cleavage sites, and venom processing enroute to the proboscis all appear to contribute to enriching the combinatorial pool of conopeptides and producing the appropriate formulation for a particular hunting situation. The complimentary tools of mass spectrometry-based proteomics and molecular biology can greatly accelerate the discovery of Conus peptides and provide insights on envenomation and other biological strategies of cone snails.

Constrained De Novo Sequencing of Peptides with Application to Conotoxins

“…Currently only about 130 mature conotoxins (meaning exact termini and modifications) are known after 40 years of study [23], so 15 novel conotoxins represents a substantial contribution to the field. (We have also observed about 35 mature conotoxins that match database sequences in C. textile, slightly exceeding the original analysis of the same data sets [36,37].) Most studies add only one or two de novo sequences at a time.…”

“…We did not obtain Rockefeller's charge-enhanced precursor data [37], only the standard carbamidomethylated cysteine. Sample preparations and data acquisition strategies were as described previously [37,36]. The Scripps data [36] were HCD and CID Orbitrap MS/MS spectra with high-accuracy precursor and fragment masses.…”

“…Sample preparations and data acquisition strategies were as described previously [37,36]. The Scripps data [36] were HCD and CID Orbitrap MS/MS spectra with high-accuracy precursor and fragment masses.…”

Various Conotoxin Diversifications Revealed by a Venomic Study of Conus flavidus

“…However, manual examination revealed that the two most intense ions were either not assigned or incorrectly assigned on the second isotopic peak. By manually analyzing the spectrum with pLabel, we found that an L28V point mutation gave a beautiful fragment ion series from b 27 ϩϩϩϩ to b 23 ϩϩϩϩ, and a T11S point mutation further led to the assignment of b12ϩϩ, b15ϩϩ, b17ϩϩ, b18ϩϩ, b19ϩϩ, and b20ϩϩϩ ions, as well as y11ϩ and y12ϩϩ ions. To match the precursor ion mass, Gly23 was mutated to Thr, which was supported by the b22ϩϩϩ ion (Fig.…”

“…Recently, MS-based proteomics techniques have come into favor for the large-scale study of conotoxin sequences. For instance, proteomic analysis of different sections of the Conus textile venom duct has been performed to explore the venom processing (23). Some elegant de novo sequencing works have also been done with conotoxins, but specialized MS methodologies were required (24,25).…”