DISULFIDE ANALYSIS WITH MASS SPECTROMETRY: TOWARD BIOTHERAPEUTIC CHARACTERIZATION

Abstract

Proteins continue to increase in use as biotherapeutic agents but need to be analyzed to verify efficacy and safety. For instance, disulfide bonds are a protein posttranslational modification (PTM) that are critically important to protein stability and function; therefore, they also impact safety and efficacy. Proteins can assume non-native bonds during recombinant expression, sample handling, or sample purification, leading to undesirable products. Currently, mapping of disulfide bonds is problematic due to sample requirements and data analysis difficulties. In this dissertation, many aspects of disulfide bonding analysis are improved. First, the fragmentation products of disulfide bonded peptides are analyzed to assist efforts in the automated analyses of these species when the analytical workflow involves LC-MS and collision-induced dissociation. Additionally, a new analysis method is introduced to more easily assign disulfide bonding partner peptides. This method utilizes a recent innovation in fragmentation, electron transfer dissociation. The method can also be used to easily detect alternate disulfide bonding patterns and confirmed the findings that there are multiple disulfide structures in a recombinant HIV glycoprotein, gp120. The method is then expanded to incorporate reporter peptides to ensure the sample preparation is not causing these alternate structures. Finally, a software tool capable of automated assignments is also outlined to expedite the data analysis. In sum, this dissertation advances the field of disulfide analysis of proteins, by introducing new methods to expedite the work and new techniques to ensure the reliability of the analyses.