Date of Award
Doctor of Philosophy (PhD)
Proteins are the building blocks of cells in living organisms, and are composed of amino acids. The expression of proteins is regulated by the processes of transcription and translation. Proteins undergo post-translational modifications in order to dictate their role physiologically within a cell.
Not all post-translational modifications are beneficial for the protein or the cell. One type of post-translational modification, called carbonylation, irreversibly places a carbonyl group onto an amino acid residue, most commonly proline, lysine, arginine, and threonine. This modification can have severe consequences physiologically, including loss of solubility, loss of function, and protein aggregation.
Carbonylated proteins have commonly been used as a marker of oxidative stress. Oxidative stress has been suggested to play a role in many human disease states, such as Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Parkinson's Disease, inflammatory diseases, and others. Evidence shows oxidative stress to be a contributing factor in the progression of aging. Therefore, markers of oxidative stress, such as carbonylated proteins, can provide key information for the development of valuable therapeutics for these conditions. However, they are found in low abundance in samples and require enrichment prior to proteomic-based studies.
Currently, affinity chromatography is the chosen method for enriching carbonylated proteins in a sample. However, the technique has significant drawbacks, including a large sample requirement, a large time requirement, the need for derivatization, and a high dilution of the sample post elution. This dissertation introduces a microfluidic enrichment technique for carbonylated proteins. The technique involves the surface modification of a polymer microchip for selective capture of carbonylated proteins. The surface chemistry is verified using different analytical techniques. Specificity of the target molecule's capture is demonstrated using a native protein. The capture conditions are optimized experimentally by studying four unique variables. Lastly, theoretical modeling is performed to determine the conditions that would lead to the technique's failure. It is seen that the technique can selectively capture target proteins from a flowing solution, even in the presence of an unoxidized protein. Protein capture is most dependent upon flow rate and crosslinker concentration. The flow rates required to break the bonds formed between an oxidized protein and the crosslinker exceeds feasible levels within a microfluidic channel. The microfluidic enrichment technique provides a promising alternative to the current "gold standard" of avidin affinity chromatography. The device has promise as a possible protein biomarker discovery tool in the search for therapeutic targets in human disease states where oxidative stress has been implicated.
Hollins, Bryant C., "" (2012). Dissertation. 298.