1 research outputs found
Evaluating Mass Spectrometry-Based Hydroxyl Radical Protein Footprinting of a Benchtop Flash Oxidation System against a Synchrotron X‑ray Beamline
Hydroxyl radical protein footprinting (HRPF) using synchrotron
X-ray radiation (XFP) and mass spectrometry is a well-validated structural
biology method that provides critical insights into macromolecular
structural dynamics, such as determining binding sites, measuring
affinity, and mapping epitopes. Numerous alternative sources for generating
the hydroxyl radicals (•OH) needed for HRPF, such as laser
photolysis and plasma irradiation, complement synchrotron-based HRPF,
and a recently developed commercially available instrument based on
flash lamp photolysis, the FOX system, enables access to laboratory
benchtop HRPF. Here, we evaluate performing HRPF experiments in-house
with a benchtop FOX instrument compared to synchrotron-based X-ray
footprinting at the NSLS-II XFP beamline. Using lactate oxidase (LOx)
as a model system, we carried out •OH labeling experiments
using both instruments, followed by nanoLC-MS/MS bottom-up peptide
mass mapping. Experiments were performed under high glucose concentrations
to mimic the highly scavenging conditions present 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 the
•OH dosage enabled optimal labeling coverage for both setups
under physiologically relevant highly scavenging conditions. Our study
demonstrates the complementarity of FOX and XFP labeling approaches,
demonstrating that benchtop instruments such as the FOX photolysis
system can increase both the throughput and the accessibility of the
HRPF technique