Date of Award
Ph.D. in Pharmaceutical Sciences
Pharmaceutics and Drug Delivery
Joshua S. Sharp
James A. Stewart
Hydroxyl radical protein footprinting (HRPF) by fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based technique that measures the apparent protein oxidation rate by freely diffusing hydroxyl radicals at different regions in a protein, and correlates changes in this apparent rate with changes in the solvent accessibility of the protein target based on quantifying the amount of oxidation of different regions of a protein. While there have been many advancements in HRPF-FPOP, oxidation measurement still suffers from high variability between measurements. High variability reduces the power statistical power this technique even in the presence of radical dosimetry measurements and controlling the quenching. The major source of this variability has not been systematically explored. Understanding the root cause of variability is crucial in improving the sensitivity and statistical power of the technique. We determined that low peptide intensity is the major source of observed variability between replicates in HRPF. We showed that the coefficients of variation of these measurements varies a lot at low signal intensity, while it stabilizes to 0.13 at high signal intensity. By simply loading more sample onto the LC column, higher peptide signal intensity was achieved, greatly reducing the variability in FPOP measurements. This method greatly increases the sensitivity of FPOP structural comparisons, an important step in applying the technique to study subtle conformational changes. While quantification of HRPF oxidation at the peptide level is relatively common and straightforward, quantification at the residue level is challenging because of the influence of oxidation on MS/MS fragmentation and only partially chromatographica
Abolhasani Khaje, Niloofar, "TOWARDS A NEW METHOD OF DETERMINING PROTEIN STRUCTURE" (2021). Electronic Theses and Dissertations. 1978.