Sequencing of T-superfamily conotoxins from Conus virgo: Pyroglutamic acid identification and disulfide arrangement by MALDI mass spectrometry

  • Amit Kumar Mandal
  • Mani Ramakrishnan Santhana Ramasamy
  • Varatharajan Sabareesh
  • Matthew E. Openshaw
  • Kozhalmannom S. Krishnan
  • Padmanabhan Balaram
Articles

Abstract

De novo mass spectrometric sequencing of two Conus peptides, Vi1359 and Vi1361, from the vermivorous cone snail Conus virgo, found off the southern Indian coast, is presented. The peptides, whose masses differ only by 2 Da, possess two disulfide bonds and an amidated C-terminus. Simple chemical modifications and enzymatic cleavage coupled with matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis aided in establishing the sequences of Vi1359, ZCCITIPECCRI-NH2, and Vi1361, ZCCPTMPECCRI-NH2, which differ only at residues 4 and 6 (Z=pyroglutamic acid). The presence of the pyroglutamyl residue at the N-terminus was unambiguously identified by chemical hydrolysis of the cyclic amide, followed by esterification. The presence of Ile residues in both the peptides was confirmed from high-energy collision induced dissociation (CID) studies, using the observation of wn- and dn-ions as a diagnostic. Differential cysteine labeling, in conjunction with MALDI-MS/MS, permitted establishment of disulfide connectivity in both peptides as Cys2-Cys9 and Cys3-Cys10. The cysteine pattern clearly reveals that the peptides belong to the class of T-superfamily conotoxins, in particular the T-1 superfamily.

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Copyright information

© American Society for Mass Spectrometry 2007

Authors and Affiliations

  • Amit Kumar Mandal
    • 1
  • Mani Ramakrishnan Santhana Ramasamy
    • 2
  • Varatharajan Sabareesh
    • 1
  • Matthew E. Openshaw
    • 3
  • Kozhalmannom S. Krishnan
    • 2
    • 4
  • Padmanabhan Balaram
    • 1
  1. 1.Molecular Biophysics UnitIndian Institute of ScienceBangaloreIndia
  2. 2.National Center for Biological SciencesBangaloreIndia
  3. 3.Shimadzu BiotechManchesterUnited Kingdom
  4. 4.Department of Biological SciencesTata Institute of Fundamental ResearchMumbaiIndia

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