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

Durand, Kirt Lenroy. Ph.D., Purdue University, December 2014. Determination of Disulfide Bond Connecting Patterns via Tandem Mass Spectrometry (MS) and Biomolecular Ion/Radical Reactions. Major Professor: Yu Xia. 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 (MS). 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