Date of Award
Chemistry and Biochemistry
Joshua S. Sharp
James V. Cizdziel
Gregory S. Tschumper
Hydroxyl radical protein footprinting (HRPF) coupled to mass spectrometry is a powerful technique for the analysis of protein topography as it generates covalent mass labels that can survive downstream sample handling, and it is sensitive to the solvent accessibility of amino acid sidechains. Of the multiple platforms for HRPF, fast photochemical oxidation of proteins (FPOP) utilizes a pulsed 248 nm KrF excimer laser to label proteins by photolyzing hydrogen peroxide. FPOP is the most widely used HRPF platform because it labels proteins faster than unfolding can occur. Variations in FPOP sample conditions make it difficult to compare results between experiments and labs. To compensate for this, reporter molecules, known as dosimeters, have been introduced to provide a metric for comparison. While several different molecules are currently in regular use, they all complicate FPOP by increasing the complexity of the sample environment and/or necessitating the addition of steps to the workflow. Here, the history of HRPF and FPOP are discussed in detail, and the development of a new dosimeter molecule, Tris(hydroxymethyl)aminomethane, is reported. This molecule is the first of its kind in that it acts as both buffer and hydroxyl radical dosimeter simultaneously, thereby significantly simplifying FPOP sample preparation. Tris acts as a gain-of-absorbance optical dosimeter as it gains absorbance at 265 nm upon oxidation, and this absorbance gain correlates well to both protein oxidation and scavenging capacity of the FPOP sample. Tris is capable of being measured in real-time through the use of an inline dosimeter which facilitates rapid adjustment of experimental parameters. Finally, a potential mechanism for Tris oxidation via reaction with hydroxyl radical is presented.
Roush, Addison, "Intrinsic Buffer Hydroxyl Radical Dosimetry for Hydroxyl Radical Protein Footprinting" (2020). Honors Theses. 1449.
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