Neurotoxic Peptides in the Multicomponent Venom of the Spider Cupiennius Salei Part II. Elucidation of the Disulphide-Bridge Pattern of the Neurotoxic Peptide CSTX-9 by Tandem Mass Spectrometry

Abstract

The disulphide-bridge pattern of the CSTX-9 polypeptide present in the multicomponent venom of the spider Cupiennius salei was determined de novo by nano-electrospray tandem mass spectrometry. Cleavage of native CSTX-9 with immobilized trypsin resulted in four disulphide-linked peptides with a mass of 2953.43 Da, which were subjected to low-energy collision-induced dissociation (CID) in a hybrid quadrupole time-of-flight (QqTOF) mass spectrometer. The product ion spectra not only provided sequence information of the peptides, the occurrence of characteristic fragment ions generated by cleavage of the peptide bonds adjacent to the bridged cysteines also allowed the disulphide-bridge pattern to be identified unambiguously. CSTX-9 was found to be a member of the neurotoxic polypeptide family incorporating the inhibitor cystine knot (ICK) structural motif. The results demonstrate the potential of modern analytical instrumentation for elucidation of complex molecular structures.