Ion trap collisional activation of disulfide linkage intact and reduced multiply protonated polypeptides.
The presence of disulfide linkages in multiply charged polypeptide ions tends to inhibit the formation of structurally informative product ions under conventional quadrupole ion trap collisional activation conditions. In particular, fragmentation that requires two cleavages (i.e., cleavage of a disulfide linkage and a peptide linkage) is strongly suppressed. Reduction of the disulfide linkage(s) by use of dithiothreitol yields parent ions upon electrospray without this complication. Far richer structural information is revealed by ion trap collisional activation of the disulfide-reduced species than from the native species. These observations are illustrated with doubly 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 of 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](11+) 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 spectra. These studies clearly suggest that the identification of polypeptides with one or more disulfide linkages via application of ion trap collisional activation to the multiply charged parent ions formed directly by electrospray could be problematic. Means for cleaving the disulfide linkage, such as reduction by dithiothreitol prior to electrospray, are therefore desirable in these cases.
Stephenson
J L
JL
Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6365, USA. stephensonjl@ornl.gov
Cargile
B J
BJ
McLuckey
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol; D007328:Insulin / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D011522:Protons
D004220:Disulfides; D007328:Insulin; D009842:Oligopeptides; D010455:Peptides; D011522:Protons; C403144:somatosin; D009113:Muramidase; D004229:Dithiothreitol