Master of Science
As humans age, exposure to oxidative stress may induce protein degradation or aggregation; both resulting in loss of protein function. Protein oxidative damage remains a dominant pathology in many common ailments. To combat these pathologies, scientists must understand the nature of oxidative modifications and their effects on protein structure and dynamics. This work employs a range of mass spectrometry (MS) methods to characterize and analyze the effects of oxidative damage on the model protein myoglobin (Mb). Mb was oxidized using tert-butyl hydroperoxide, and the resulting modifications were characterized by top-down and bottom-up MS workflows. Hydrogen/deuterium exchange MS indicated elevated structural dynamics in oxidatively modified regions. Collision-induced activation showed that oxidized Mb loses heme more readily than its unmodified counterpart. Surprisingly, ion mobility experiments uncovered that collision-induced unfolding produces more compact non-native gas phase structures for the oxidized protein. The methods applied provide an analytical foundation for the comprehensive characterization of oxidative damage that will be applicable to many other proteins.
Summary for Lay Audience
Oxidative damage is the result of an overabundance of reactive oxygen-containing chemicals in the body. These chemicals arise as the result normal cellular processes but can be exacerbated by external factors like smoking or tanning. Antioxidants in the body combat the effects of these chemicals, but an imbalance can result in damaging reactions occurring inside of the body (i.e., oxidative damage). As living things age, deterioration of biological processes can be partially attributed to accumulation of oxidatively modified proteins – the biological building block for cells and tissues. Oxidative damage has been implicated as one of the causes of common ailments including glaucoma and neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease. Looking into the eyes of someone who suffers glaucoma, the direct result of oxidative protein damage can be seen. The cloudiness in the eye is produced by the accumulation of proteins that have lost function due to extensive oxidation, unrepairable by the body.
This work aims to examine the factors that lead to the disfunction of proteins after oxidative damage. Working with oxidized proteins poses difficulty because they tend to cause problems for many conventional analytical methods; one of these difficulties is their non-uniform mass distribution. Here, we developed a procedure that mimics the effects of reactive oxygen species in the body. Mass spectrometry (MS), a method involving the characterization of gaseous protein ions produced by electrospray ionization, proved to be a versatile approach that allowed us to accurately assess modifications. By applying various MS methods, we were able to determine the chemical nature of modifications, their locations, and their effects on protein structure and dynamics. In this way it was possible to obtain valuable information that might one day help with the development of treatments to combat oxidative damage. Earlier MS studies have produced valuable information on oxidized proteins but neglected certain methods due to onerous data analysis. The current work addresses many of these difficulties, thereby establishing a comprehensive MS-based platform for the characterization of oxidatively damaged proteins.
Saullo, Vincent, "Analysis of Oxidatively Damaged Proteins by Mass Spectrometry" (2020). Electronic Thesis and Dissertation Repository. 6823.