Sequencing and mass profiling highly modified conotoxins using global reduction/alkylation followed by mass spectrometry.
A novel high-throughput method for characterizing heavily modified peptides from cone snail venom is described. Unpurified cone snail duct venom, consisting primarily of multiply disulfide-bonded peptides, is reduced and alkylated using a global procedure in order to simultaneously reduce and derivatize dozens of disulfide-bonded peptides. Samples of Conus victoriae venom are analyzed by online liquid chromatography-electrospray ionization-ion trap-mass spectrometry (LC-ESI-MS) with collisionally induced dissociation (CID). Comparison of the mass profiles of peptides and CID spectra before and after the global reduction and alkylation enables cysteine-containing conopeptides to be ascertained. In this case, over 40 conotoxins are characterized based on only two LC-ESI-MS experiments in terms of mass, number of disulfide-linked cysteine residues (and hence, potential toxin superfamilies), relative hydrophobicity, and other posttranslational modifications. Using this technique, over half of the amino acids (by mass) of several peptides are defined prior to any detailed sequencing studies. Further comparison of the mass data with previously published genetic information allows sequence verification of three novel peptides, termed vc5b, vc6b and vc6c, based on both LC-ESI-MS CID and nanoelectrospray ionization-ion trap-mass spectrometry (nanoESI-MS) experiments. This global method is ideally suited to the use of larger genetic databases in order to efficiently sequence peptides in Conus venoms and is also applicable to analysis of other disulfide-rich classes of peptides such as defensins, chemokines, and snake, spider, or other venoms.
Jakubowski
Jennifer A
JA
Department of Chemistry and the Beckman Institute, University of Illinois, Urbana, Illinois 61801, USA.
Sweedler
Jonathan V
JV
D000478:Alkylation; D000595:Amino Acid Sequence; D020916:Conotoxins / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D036103:Nanotechnology; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational
D020916:Conotoxins