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Abstract
Hydroxyl radical protein footprinting (HRPF) is an emerging structural technique with its advantage of providing a fast, relatively nonspecific, covalent label that probes a variety of solvent accessible amino acids within one experiment. Development of an HRPF protocol to generate constrained, accurate computational models would have large impact on numerous biological targets that have remained resistant to high-resolution structural interrogation. Previous work has shown that an electron-transfer dissociation (ETD) - based HRPF method can be successfully used to probe protein structure and dynamics. However, little work has been done for absolute protein topography measurements. An optimized HRPF protocol that can help to better interpret and understand HRPF data as well as provide information on their relationship with biophysical properties while at the same time improve the ease of HRPF data collection and analysis would be preferred. The work presented describes a new method using size exclusion chromatography coupled with ETD for relative quantification of sites of peptide and protein modification for easy data collection and analysis, as well as the development of an adenine dosimetry system for HRPF experiment use to monitor the effective amount of hydroxyl radical delivered into each biological system in order to compare HRPF data between samples. This work also reported using multi-point FPOP technology with combination of adenine dosimetry system for measuring absolute protein topography by HRPF for future HRPF topography-constrained modeling.