Determination of disulfide bond connecting patterns via tandem mass spectrometry (MSn) and biomolecular ion/radical reactions

Disulfide bond formation is one of the most common post translational modifications to occur in proteins and naturally occurring peptides. Disulfide bond formation plays a critical role in stabilizing their three-dimensional structure; therefore, it is very important to pinpoint the correct disulfide bond connecting pattern in order to fully understand the biological functions of these proteins and peptides. To fully characterize an analyte containing disulfide bonds, the sequence must first be known followed by the disulfide bond connecting pattern. This presents an analytical challenge as there are very few methodologies that can produce those essential pieces of information. The gold standard for analyzing these disulfide linked analytes is to enzymatically digest them, separate them via high performance liquid chromatography (HPLC) and then analyze them via tandem mass spectrometry (MSn). Although effective, the enzymatic digestion approach can be expensive, time consuming, nonspecific (some enzymes have a broad range of specificity), and sometimes initiate disulfide bond scrambling. The goal of the research in this dissertation is to develop novel gas-phase methodologies for determining how disulfide bonds are connected (as well as enhanced sequence coverage) in regio-isomeric peptides containing two intramolecular disulfide bonds. Two novel methods were successfully used to identify disulfide bond connecting patterns as well as produce enhanced sequence coverage. The first method utilizes MS n of internal fragment ions. MS2 collision induced dissociation (CID) of intact peptide ions produced internal fragment ions from a region of the peptide covered by a disulfide bond. The formation of an internal fragment ion converted an intrachain disulfide bond to an interchain disulfide bond. MS3 CID of the internal fragment ion produced unique b and y ions that accurately identified the disulfide bond connecting pattern. The second methodology used ion/radical reactions and takes advantage of the disulfide bond\u27s susceptibility to radical attack. The methoxy radical was generated via UV photolysis and allowed to react with disulfide linked regio-isomeric peptides. MS2 CID was conducted on the reaction product corresponding to the cleavage of one disulfide bond. MS3 CID was conducted on y ions that contained three cysteine residues and one intact disulfide bond. Unique b and y ions that accurately identified the disulfide bond connecting pattern were produced. The latter methodology was also applied to the analysis of bovine insulin (two intermolecular and one intramolecular disulfide bond) to show the practicality in the analysis of complex analytes