title: Probing peptide libraries from Conus achatinus using mass spectrometry and cDNA sequencing: identification of delta and omega-conotoxins.
abstract: The peptide library present in the venom of the piscivorous marine snail Conus achatinus has been probed using a combination of mass spectrometry and cDNA sequencing methods. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis, before and following global reduction/alkylation of peptide mixtures, permits the rapid classification of individual components on the basis of the number of disulfide bonds. Mass fingerprinting and the reverse phase HPLC retention times permit a further deconvolution of the library in terms of peptide size and hydrophobicity. Sequencing of cDNA derived using O-superfamily specific primers yielded five complete conotoxin precursor sequences, ranging in polypeptide length from 75-87 residues containing six Cys residues at the C-terminus. Sequence analysis permits classification of the five putative mature peptides (Ac 6.1 to Ac 6.5) as delta, omega, and omega-like conotoxins. The presence of these predicted peptides in crude venom was established by direct matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) sequencing following trypsin digestion of the peptide mixture after global reduction/alkylation. The determination of partial peptide sequences and comparison with the predicted sequences resulted in the identification of four of the five predicted conotoxins. The characterization of posttranslationally modified analogs, which are hydroxylated at proline or amidated at the C-terminus is also demonstrated. Crude venom analysis should prove powerful in studying both inter- and intra-species variation in peptide libraries.
authors.affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India.
authors.forename: Konkallu Hanumae
authors.initials: KH
authors.lastname: Gowd
authors.forename: Kalyan Kumar
authors.initials: KK
authors.lastname: Dewan
authors.forename: Prathima
authors.initials: P
authors.lastname: Iengar
authors.forename: Kozhalmannom S
authors.initials: KS
authors.lastname: Krishnan
authors.forename: Padmanabhan
authors.initials: P
authors.lastname: Balaram
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020916:Conotoxins / Q000737:chemistry*; D052078:Conus Snail / Q000737:chemistry*; D018076:DNA, Complementary / Q000378:metabolism*; D008969:Molecular Sequence Data; D019151:Peptide Library; D017422:Sequence Analysis, DNA / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D020960:omega-Conotoxins / Q000737:chemistry
chemical_list: D020916:Conotoxins; D018076:DNA, Complementary; D019151:Peptide Library; D020960:omega-Conotoxins