The sensitivity of mass spectrometry combined with the separatory power of high-performance liquid chromatography was used to investigate the venom of individual cone shells. This analysis has revealed that cone venoms contain a complex mixture of peptides which vary quantitatively and qualitatively both between and within species. A differential alkylation procedure followed by tandem mass spectrometric analysis can be used to determine the disulfide bond connectivity in these small, cysteine-rich peptides.
Conidae are a family of venomous marine gastropods ( -500 species) that are common in tropical and subtropical waters. Venom is comprised of mostly small, highly constrained, multiply disulfide-bridged peptides which are used in conjunction with harpoon-like radular teeth to rapidly immobilize prey. These venoms have yielded a wide variety of novel bioactive peptides which act selectively at a number of sites, including sodium and calcium channels and nicotinic acetylcholine receptors.'. * Analysis of these venoms to date has been facilitated by reverse phase highperformance liquid chromatography (HPLC).'. * LC/MS analysis potentially allows further insights into the complexity of the venoms.
Disulfide bonds between cysteine residues are one of the most important means of inducing stability in protein conformations3 and to maintain the tertiary structure of small peptides (mini-proteins) and hence promote specific receptor interactions. The determination of cysteine connectivity is of importance for both functional and structural studies. Disulfide bond connectivities have typically been investigated by a number of methods including Edman ~equencing~-~ and high energy, collision-induced dissociationlmass spectrometry (CID/MS).*-l' Recent papers by Sun and Chang" and Gray'' have demonstrated the feasibility of connectivity determination using reduction alkylation procedures combined with HPLC separation and Edman sequencing.
Ion-spray mass spectrometry can be used to identify and characterize peptides both before and after cysteine modification. Molecular weights of peptides and peptide mixtures may be determined directly, using on-line HPLC/ MS. In addition, mass spectrometry can identify post-translationally modified amino acids and provide strong evidence for homogeneity of isolated peptides. However, further characterization is often necessary for structural identification and amino acid sequencing. Tandem mass spectrometry (MS/MS) is a powerful technique for determining sequence, with the advantage over classical Edman degradation of not being limited to peptides with unblocked N-termini and it is well suited to the detection of novel post-translationally modified amino acids, a common