Disulfide Bond Assignments by Mass Spectrometry of Native Natural Peptides: Cysteine Pairing in Disulfide Bonded Conotoxins

摘要

The critical, and often most difficult, step in structure elucidation of diverse classes of natural peptides is the determination of correct disulfide pairing between multiple cysteine residues. Here, we present a direct mass spectrometric analytical methodology for the determination of disulfide pairing. Protonated peptides, having multiple disulfide bonds, fragmented under collision induced dissociation (CID) conditions and preferentially cleave along the peptide backbone, with occasional disulfide fragmentation either by C~(beta)-S bond cleavage through H~(alpha) abstraction to yield dehydroalanine and cysteinepersulfide, or by S-S bond cleavage through H~(beta) abstraction to yield the thioaldehyde and cysteine. Further fragmentation of the initial set of product ions (MS~(n)) yields third and fourth generation fragment ions, permitting a distinction between the various possible disulfide bonded structures. This approach is illustrated by establishing cysteine pairing patterns in five conotoxins containing two disulfide bonds. The methodology is extended to the Conus araneosus peptides Ar1446 and Ar1430, two 14 residue sequences containing 3 disulfide bonds. A distinction between 15 possible disulfide pairing schemes becomes possible using direct mass spectral fragmentation of the native peptides together with fragmentation of enzymatically nicked peptides.