Browsing Theses and Dissertations School of Pharmacy by Title "Development of Fast Photochemical Oxidation of Proteins for in Vivo Modification in Caenorhabditis elegans"
Now showing items 1-1 of 1
Development of Fast Photochemical Oxidation of Proteins for in Vivo Modification in Caenorhabditis elegansMass spectrometry (MS) has become widely used for the characterization of protein structure and protein-protein interactions (PPI). Unlike many commonly used structural methods, MS is not limited by the size of molecules, thus allowing for the study of a wide range of purified protein complexes, cells, tissues, and complex organisms. Instrumentation advancements have also decreased the need for large sample concentrations and have increased mass accuracy and resolution. In the past decade, MS-based protein footprinting has become increasingly utilized for the determination of higher-order protein structure and provides residue-level analysis on PPI interaction sites, protein-ligand interactions, and regions of conformational change by covalently modifying the solvent-accessible surface area (SASA) of proteins through the use of a small chemical label. The hydroxyl radical protein footprinting (HRPF) method, fast photochemical oxidation of proteins (FPOP), utilizes hydroxyl radicals (•OH) to oxidatively modify solvent-accessible amino acid side chains. These radicals are generated via hydrogen peroxide photolysis using a KrF excimer laser at a 248 nm wavelength. To date, most applications of FPOP have been performed in vitro in relatively pure protein systems. Most notably, it has been applied for antibody epitope mapping, protein folding, and protein aggregation. This work focuses on the extension of FPOP for in vivo protein structural analysis in Caenorhabditis elegans, a method entitled in vivo FPOP (IV-FPOP). FPOP is particular suited for in vivo protein studies because of the irreversible nature of the modification, which mitigates time constraints with respect to sample preparation, proteomic digestion, and sample processing. Additionally, the •OH generated can label 19 out of 20 amino acids allowing for the study of multiple proteins regardless of protein sequence or cellular location. Given the complexity of the platform, numerous parameters required optimization for maximum labeling efficiency including the development of a microfluidic flow system for the labeling of worms by IV-FPOP, hydrogen peroxide concentration optimization, and the addition of chemical penetration enhancers to increase hydrogen peroxide uptake by the worm, and the implementation of a multiplexing proteomics platform increased throughput of IV-FPOP oxidatively modified peptides.