DOI
https://doi.org/10.25772/A138-TH48
Defense Date
2015
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Chemistry
First Advisor
Dr. Scott Gronert
Abstract
Oxidative stress can result in changes to many biomolecules and also affect their activities. We are interested in protein carbonylation, a type of unnatural oxidation which has been associated with numerous degenerative disease states and is also a consequence of the natural aging process. Protein carbonyls are stable species, but countless analytical barriers exist in terms of their identification. Thus, the main goal of this work was to develop and optimize analytical methods that could be used to help us better understand which, where, and how proteins are being carbonylated.
Initial studies involved method validation for carbonylating, tagging, and enriching the model protein human serum albumin (HSA). We have developed a reproducible method of producing carbonylated protein in vitro in which HSA is treated with acrolein to carbonylate cysteines, histidines, and lysines. Protein carbonyls are compatible with various affinity labels and enrichment techniques. We strived to learn more about the efficiencies of various biotin affinity labels and avidin enrichment techniques using quantitative assays and mass spectrometry. Results showed a preference for different affinity labels based on their chemical properties and suggested that monomeric columns are selective for particular peptides. Most recently, method development and validation work was done involving a cleavable biotin tag that enables both enrichment and identification of protein carbonylation modification sites. This affinity tag offered the highest labeling efficiency of all tags tested in the past and greater coverage of modification sites than biotin hydrazide reagents.
We applied our analytical methods to two sets of human blood samples. The first sample set was plasma taken from chronic kidney disease (CKD) patients. No carbonylation patterns were elucidated, but this project marked the beginning of blood analyses in which existing protocols were adapted to blood samples. The second sample set was serum/plasma taken from patients with traumatic injuries. We effectively applied our analytical methods to these sample sets and were able to visualize and quantitate temporal protein carbonylation patterns via Western blotting and iTRAQ-based mass spectrometry experiments. ProteoMiner experiments proved successful in that we were able to identify a larger and more diverse amount of carbonylated proteins via mass spectrometry.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-6-2015