Characterisation of Conus venom using polypeptide separation methods and mass spectrometric analysis
Each of the 500 known Conus species has its own distinctive, complex and peptide rich venom. Approximately 100 different peptide toxins, so called conotoxins can be expressed with little similarity between the species. An overwhelming majority of conotoxins are probably targeted selectively to specific ion channels. Because conotoxins can discriminate between closely related subtypes of ion channels, many of the characterised conotoxins are used as pharmacological agents in ion channel research. Several have direct diagnostic and therapeutic potential. Although the venoms have been studied for the last two decades, only a miniscule fraction of the postulated conotoxins have been identified. The studies presented in this dissertation were set out with the aim to further characterise the relative unknown venom of Conus ixntricosus and to explore the protein content of Conus venoms in general. In total five different venoms were investigated. Chapter three describes the analysis of protein content in various Conus venoms. Although both one and two-dimensional gel electrophoresis was applied, no known toxin precursors were found. Proteins identified are mainly housekeeping proteins, such as actin. One of the strategies applied for finding novel conotoxins involved a multi dimensional liquid chromatography experiment where a trypsin digest was performed in between two separations before mass spectrometric analysis. One putative novel partial conotoxin was found. Chapters five and six describe the mass spectrometric analysis of Conus ventricosus and textile venom after a two-dimensional liquid chromatography separation. With the methods applied, besides verifying a number of putative sequences, several novel conotoxins were found, in particular a number of so called contryphans. One of these contryphans found contained a number of post-translational modifications, amongst them hydroxyproline and the very rare brominated tryptophan.