Dissociations of disulfide-linked gaseous polypeptide/protein anions: ion chemistry with implications for protein identification and characterization.

Ion trap collisional activation of whole protein anions that contain disulfide bonds results in the cleavage of one of the bonds that comprises the disulfide linkage. The disulfide linkage can break at any of three possible locations, giving rise to several products with different partitioning of sulfur atoms. A facile second-generation dissociation occurs at the polypeptide backbone from products formed from cleavage of the nearest C-S bond of a disulfide linkage. This cleavage occurs exclusively at the N-terminal side of the cysteine residue, from which the C-S bond was cleaved, thereby yielding c and z-S type product ions. This secondary reaction is apparently a relatively low-energy reaction with relatively high entropy requirements because it is not observed to be a major process under beam-type collisional activation conditions, but is a major process under ion trap collisional activation conditions. The specificity of this cleavage, as well as the ability to distinguish it from other cleavages by the sulfur atom distribution, make it useful for the identification of unknown proteins via database searching. Furthermore, the pattern of disulfide cleavages can be useful in providing information about the location of post-translational modifications. Examples using bovine pancreatic trypsin inhibitor and ribonuclease A and B are given to illustrate these points.