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Abstract
Hydroxyl radical protein footprinting (HRPF) using synchrotron X-ray radiation and mass spectrometry is a well-validated structural biology method that is providing critical insights into macromolecular dynamics. Numerous alternative sources for HRPF such as laser photolysis and plasma irradiation complement synchrotron-based HRPF. A recently developed commercially available instrument based on flash lamp photolysis, the Fox® system, enables access to laboratory benchtop HRPF. Here, we evaluate the feasibility of standardizing HRPF experiments in-house with a benchtop Fox® instrument as a precursor to synchrotron-based X-ray footprinting at the NSLS-II XFP beamline. Using lactate oxidase enzyme (LOx) as a model system, we carried out hydroxyl radical (•OH) labeling experiments using both instruments, followed by nanoLC-MS/MS bottom-up peptide mass mapping. Experiments were performed with high glucose concentrations to mimic highly scavenging conditions in biological buffers and human clinical samples, where less •OH are available for reaction with the biomolecule(s) of interest. The performance of the Fox® and XFP HRPF methods was compared, and we found that tuning •OH dosage enabled an optimum labeling coverage for both setups under physiologically relevant highly scavenging conditions. Our study demonstrates the complementarity of Fox® and XFP labeling approaches, showing that benchtop instruments such as Fox® photolysis system can increase throughput and accessibility of HRPF technology.
Supplementary materials
Title
Evaluating mass spectrometry based hydroxyl radical protein footprinting of a benchtop Flash oxidation system against a synchrotron X-ray beamline
Description
Increase in glucose concentrations scavenges hydroxyl radicals and reduces decay of internal dosimeter fluorescence/UV absorbance (Figure S1, S-2), trypsin digestion of LOx revealed similar labeling coverage on XFP and Fox® platforms (Figure S2, S-3), hydroxyl modification of selected LOx peptides on Fox® platform in physiological pH and scavenging conditions vs. adenine absorbance (Figure S3, S-4), effect of different post-labeling quenching conditions in FOX® platform for LOx peptides (Figure S4, S-5), hydroxyl modification rates of selected LOx peptides on Fox® platform in physiological pH and scavenging conditions vs. adenine absorbance (Table S1, S-6).
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