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Probing peptide libraries from Conus achatinus using mass spectrometry and cDNA sequencing: identification of δ and ω‐conotoxins
Author(s) -
Gowd Konkallu Hanumae,
Dewan Kalyan Kumar,
Iengar Prathima,
Krishnan Kozhalmannom S.,
Balaram Padmanabhan
Publication year - 2008
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.1377
Subject(s) - chemistry , conotoxin , peptide , mass spectrometry , tandem mass spectrometry , bottom up proteomics , cdna library , matrix assisted laser desorption/ionization , chromatography , peptide sequence , complementary dna , protein mass spectrometry , biochemistry , desorption , gene , organic chemistry , adsorption
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 δ, ω, and ω‐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. Copyright © 2008 John Wiley & Sons, Ltd.