Ion trap collisional activation of disulfide linkage intact and reduced multiply protonated polypeptides

The presence of disulfide linkages in multiply charged polypeptide ions ten ds to inhibit the formation of structurally informative product ions under conventional quadrupole ion trap collisional activation conditions, In part icular, fragmentation that requires two cleavages (i.e., cleavage of a disu lfide linkage and a peptide linkage) is strongly suppressed, Reduction of t he disulfide linkage(s) by use of dithiothreitol yields parent ions upon el ectrospray without this complication. Far richer structural information is revealed by ion trap collisional activation of the disulfide-reduced specie s than from the native species. These observations are illustrated with dou bly protonated native and reduced somatosin, the [M + 5H](5+) ion of native bovine insulin and the [M + 4H](4+) and [M + 3H](3+) ions of the B-chain o f bovine insulin produced by reduction of the disulfide linkages in insulin , and the [M + 11H](11+) ion of native chicken lysozyme and the [M + 11H](1 1+) and [M + 14H](14+) ions of reduced lysozyme, In each case, the product ions produced by ion trap collisional activation were subjected to ion/ion proton transfer reactions to facilitate interpretation of the product ion s pectra, These studies clearly suggest that the identification of polypeptid es with one or more disulfide linkages via application of ion trap collisio nal activation to the multiply charged parent ions formed directly by elect rospray could be problematic, Means for cleaving the Bisulfide linkage, suc h as reduction by dithiothreitol prior to electrospray, are therefore desir able in these cases, Copyright (C) 1999 John Whey & Sons, Ltd.