Advancing the Applicability of Fast Photochemical Oxidation of Proteins to Complex Systems

Abstract

Hydroxyl radical protein footprinting coupled with mass spectrometry has become an invaluable technique for protein structural characterization. In this method, hydroxyl radicals react with solvent exposed amino acid side chains producing stable, covalently attached labels. Although this technique yields beneficial information, the extensive list of known oxidation products produced increases the complexity of identifying and quantifying oxidation products. The current methods available for quantifying the extent of oxidation either involve manual analysis steps, or limit the number of searchable modifications or the size of sequence database. This creates a bottleneck which can result in a long and arduous analysis process, which is further compounded in a complex sample. In addition to the data complexity, the peptides containing the oxidation products of hydroxyl radical-mediated protein footprinting experiments are typically much less abundant than their unoxidized counterparts. This is inherent to the design of the experiment as excessive oxidation may lead to undesired conformational changes or unfolding of the protein, skewing the results. Thus, as the complexity of the systems studied using this method expands, the detection and identification of these oxidized species can be increasingly difficult with the limitations of data-dependent acquisition (DDA) and one-dimensional chromatography. The recently published in cell FPOP method exemplifies where this field is headed - larger and more complex systems. This dissertation describes two new methodologies and one new technology for hydroxyl radical-mediated protein footprinting, expanding the applicability of the method. First is development of a new footprinting analysis method for both peptide and residue level analysis, allowing for faster quantification of results. This method utilizes a customized multilevel search workflow developed for an on-market search platform in conjunction with a quantitation platform developed using a free Excel add-in, expediting the analysis process. Second is the application of multidimensional protein identification technology (MudPIT) in combination with hydroxyl radical footprinting as a method to increase the identification of quantifiable peptides in these experiments. Last is the design and implementation of a flow system for in cell FPOP, which hydrodynamically focuses the cells, and when used yielded a 13-fold increase in oxidized proteins and 2 orders of magnitude increase in the dynamic range of the method.