FAST ATOM BOMBARDMENT MASS SPECTROMETRY OF DISULFIDE CONTAINING PEPTIDES AND PROTEINS
Abstract
An investigation was conducted to determine disulfide bond linkage in peptides and proteins using fast atom bombardment mass spectrometry (FABMS). Both intra- and interchain disulfide-containing peptides have been the subject of this investigation. The characterization of disulfide bonds in peptides with FABMS was studied by exposing an acidic solution of a disulfide-containing peptide in a matrix, such as a mixture of dithiothreitol (DTT) and dithioerythritol (DTE) and/or glycerol, to high energy xenon atom beam for an extended time (less than 15 minutes). The results indicated the reduction of disulfide bonds in peptides occurred during FABMS analysis and is independent of the type of matrix used. A technique for assignment of disulfide bonds in peptides and proteins, which is based on reduction of disulfide bonds either directly during FABMS analysis or indirectly by chemical reduction followed by FABMS analysis, has been developed. The method for characterization of interchain disulfide bonds is based on the increase in the intensity of the molecular ions of the reduced peptides with a simultaneous decrease in the intensity of the molecular ion of the oxidized peptide. This information assists one to identify peptide fragments covalently linked via intermolecular disulfide bonds. The intrachain disulfide bonds are, on the other hand, identified by the increase in the intensity of the molecular ion of the reduced peptide, relative to the intensity of the molecular ion of the oxidized peptide. Time-dependent FABMS analysis of a series of synthetic peptides with and without disulfide bonds indicated that detectable amounts of new compound(s) formed by new cross-linkages are not produced. Such new compounds could conceivably be produced during the course of digestions and/or prolonged FABMS analysis and would lead to an incorrect structure assignment to the initial peptide. In addition, FABMS analysis of the tryptic digests of CNBr-treated hen egg-white lysozyme and bovine ribonuclease A showed that complete assignment of disulfide bonds in a protein may be impossible without prior chromatographic purification of some of the peptides having the disulfide bond(s).
Degree
Ph.D.
Subject Area
Biochemistry
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