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We have isolated and characterized ␣-conotoxin EpI, a novel sulfated peptide from the venom of the molluscivorous snail, Conus episcopatus. The peptide was classified as an ␣-conotoxin based on sequence, disulfide connectivity, and pharmacological target. EpI has homology to sequences of previously described ␣-conotoxins, particularly PnIA, PnIB, and ImI. However, EpI differs from previously reported conotoxins in that it has a sulfotyrosine residue, identified by amino acid analysis and mass spectrometry. Native EpI was shown to coelute with synthetic EpI. The peptide sequence is consistent with most, but not all, recognized criteria for predicting tyrosine sulfation sites in proteins and peptides.

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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 Author for correspondence. feature in conopeptides.2 The singly or multiply charged molecular ions from linear peptides typically fragment by cleavage of the peptide backbone under CID conditions. The majority of fragment (product) ions observed under low-energy conditions (typically 20-40 V for a quadrupole mass spectrometer)...

We previously reported an efficient peptide synthesis method, AJIPHASE ,t hat comprises repeated reactions and isolations by precipitation. This method utilizes an anchor molecule with long-chain alkyl groups as ap rotecting group for the C-terminus.T of urther improve this method, we developed ao ne-pot synthesis of ap eptide sequence wherein the synthetic intermediates were isolated by solvent extraction instead of precipitation. Ab ranched-chain anchor molecule was used in the new process,s ignificantly enhancing the solubility of long peptides and the operational efficiency compared with the previous method, whiche mployed precipitation for isolation and as traight-chain aliphatic group. Another prerequisite for this solvent-extraction-based strategy was the use of thiomalic acid and DBUfor Fmoc deprotection, which facilitates the removal of byproducts,such as the fulvene adduct.

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