Corie Y. Ralston

Protein oxidative footprinting, using hydroxyl radical labeling detected by bottom-up proteomics, has progressed from an emerging method to a widely used approach in structural biology. Hydroxyl radicals generated from hydrogen peroxide (via photolysis, Fenton chemistry or electrochemistry) or directly from water (via X-rays, plasma or gamma rays) irreversibly encode structural information within protein side chains, which is read out using standard liquid chromatography-mass spectrometry workflows. Quantitative changes in labeling report on solvent accessibility and reveal effects of protein-protein interactions, ligand binding, protein folding, conformational changes or applied stress. Comparing labeling patterns between states provides detailed maps of structural changes and interaction sites. Over the past decade, oxidative footprinting has proven valuable as a solution-phase and in-cell method for protein structure analysis. This Perspective summarizes best practices for experimental design, sample processing, data analysis, interpretation and integration with orthogonal data, offering a consensus framework to guide application of oxidative footprinting in academic and biopharmaceutical research.

The structural biology method of X-ray footprinting mass spectrometry (XFMS) is available at two national synchrotron beamlines in the USA: one at the Advanced Light Source (ALS) on the West Coast and the other at the National Synchrotron Light Source II on the East Coast. XFMS is a solution-state technique that utilizes oxidative modifications of proteins at micromolar concentrations in aqueous buffer to extract structural information. X-rays are employed to generate hydroxyl radicals in situ , which covalently modify specific protein side chains. These modifications are subsequently quantified using liquid chromatography and mass spectrometry. Ratiometric changes in modification levels between two protein states ( e.g. with and without ligand) generate a relative solvent accessibility map of the protein pairs, which serves to reveal structural features. Up until recently, the XFMS capability was available as part of a shared program at the ALS without a dedicated beamline. In this article, we describe the commissioning of ALS beamline 3.3.1, dedicated to XFMS, including the installation of a new focusing mirror, the design and construction of a new endstation with automated sample handling and exposure capabilities, and the use of accurate empirical dose calculations using Gafchromic film. Finally, we showcase the new beamline capabilities using two protein systems.

Synchrotron radiation (SR) sources provide unparalleled brilliance, collimation, coherence, and tunability, enabling specialized techniques that are crucial for advancing medical research across diverse fields from radiation oncology to rational drug design. Certain SR methods, such as macromolecular crystallography, are highly developed and automated, and have been used for decades for both fundamental understanding of biomolecules as well as pharmaceutical design, while other methods, such as microbeam radiation therapy, represent relatively recent developments. Scattering and diffraction methods using SR can provide atomic-level structural mapping of proteins, nucleic acids, and complexes. Imaging applications using SR continue to be developed and advanced for mapping of biological structures and potential use as diagnostics in disease detection. Spectroscopic methods are used to study elemental distributions relevant for detection of contamination in biological systems. Collectively, SR research delivers detailed multiscale structural and molecular information essential for addressing complex biomedical challenges. In this mini-review, we cover SR methods that are specifically relevant to medical applications, focusing mainly on SR X-ray and SR infrared techniques and applications. We include diffraction and scattering, imaging, absorption and fluorescent spectroscopy, use of SR in oncology treatments, and radiation damage methods that integrate one or another aspect of an SR method in conjunction with other non-SR techniques.

The method of X-ray footprinting and mass spectrometry (XFMS) using high flux synchrotron X-ray sources has become an established method in structural biology and is based on the radiolytic production of hydroxyl radicals, which oxidatively modify protein sidechains. While other methods of producing hydroxyl radicals are available, one benefit of using high flux density sources is that hydroxyl radical scavenging reactions can be minimized, and exposure times kept short to minimize secondary reactions. Here we present an application of the XFMS method using low dose rate X-rays from a commercial instrument. We demonstrate the feasibility of the approach using short peptides, characterizing the oxidative modifications +14, +16, and +32 Da under both aerobic and low oxygen conditions, and we additionally quantify the hydrogen peroxide production for various doses using the low dose rate source. These results provide fundamental information on the oxidative damage to peptides due to hydroxyl radicals using a low dose rate X-ray source.

The method of X-ray Footprinting and Mass Spectrometry (XFMS) using high brightness synchrotron X-ray sources has become an established method in structural biology and is based on the radiolytic production of hydroxyl radicals which oxidatively modify protein sidechains. While other methods of producing hydroxyl radicals are available, one benefit of using high flux density sources is that hydroxyl radical scavenging reactions can be minimized, and exposure times kept short to minimize secondary reactions. Here we present an application of the XFMS method using low dose rate X-rays from a commercial instrument. We demonstrate the feasibility of the approach using short peptides, characterizing the oxidative modifications +14, +16, and +32 Da under both aerated and low-oxygen conditions, and we additionally quantify the hydrogen peroxide production for various doses using the low dose rate source. These results provide fundamental information on the oxidative damage to peptides due to hydroxyl radicals using a low dose rate X-ray source.

Organophosphorus chemical warfare agents (CWAs) pose urgent threats to human health and the environment, driving the search for highly efficient catalytic detoxification strategies. Zirconium based metal-organic frameworks (Zr-MOFs) have long been regarded as the most effective catalysts for CWA simulant hydrolysis, owing to the open Lewis acidic sites of their robust Zr6-oxo clusters. This performance established MOF-808 as the accepted state of the art and has remained unsurpassed for the past decade. The Zr6 clusters in MOFs that enable high activity also bind products too strongly, which poisons active sites and restricts turnover, creating an apparent performance ceiling. Herein, we introduce a generalizable node-engineering strategy that achieves Nb substitution of Zr within canonical Zr6 clusters, creating mixed metal Zr6-xNbx clusters rarely realized in MOFs. This approach is demonstrated across four archetypal Zr-MOFs (UiO-66, UiO-68, MOF-808, NU-1000) while preserving framework integrity and porosity. The resulting Zr/Nb-MOFs accelerate phosphoester hydrolysis by up to two orders of magnitude, with MOF-808-Zr/Nb delivering the fastest rates reported to date. Under continuous-flow operation, MOF-808-Zr/Nb maintains superior catalytic activity and durability at concentrations down to 1 ppm. These results establish Nb node engineering as a generic approach for creating next generation heterogeneous catalysts in environmental remediation and chemical defense.

ALS-ENABLE is an integrated NIH P30 resource at the Advanced Light Source synchrotron at Lawrence Berkeley National Laboratory in Berkeley, California, USA. The resource provides a single portal to the combined mature structural biology technologies of macromolecular crystallography, small-angle X-ray scattering and X-ray footprinting mass spectrometry, and includes beamlines 2.0.1, 3.3.1, 4.2.2, 5.0.1, 5.0.2, 5.0.3, 8.2.1, 8.2.2, 8.3.1 and 12.3.1. This paper describes the organizational structure and the technologies of ALS-ENABLE. A case study showcasing the main technologies of the resource applied to the characterization of the SpyCatcher–SpyTag protein system is presented.

Alzheimer's disease has long been associated with the aggregation of amyloid beta peptides (Aβ42) into macroscale plaques, although specific neurodegenerative agents have not been definitively identified. Much evidence has pointed to the soluble nanoscale oligomers that form early in the Aβ42 aggregation pathway, but there is little understanding of these structures, their mechanisms of formation, or how they grow into plaques. Here, we show that a solvatochromic fluorophore with near-infrared (NIR) emission can track synthetic Aβ42 aggregation through environment-sensitive spectral shifts from the earliest time points through plaque formation. This azide-functionalized phosphine oxide azetidine rhodol (Phazr-N₃) shows large polarity-dependent changes in fluorescence emission, with maxima shifting from 630 nm in toluene to 703 nm in aqueous buffer, and a maximum quantum yield of 62%. Upon induction of Aβ42 aggregation, we observe immediate solvatochromic changes in Phazr-N₃ fluorescence, with multiple apparent phases over 12 h, and which culminate before the onset of any major fluorescence changes of conformation-specific aggregation fluorophore thioflavin T. Solution anisotropy measurements show a low micromolar affinity of Phazr-N₃ for disordered, free Aβ42 in solution, and real-time measurements are consistent with rapid liquid-liquid phase separation and slow dehydration of the growing aggregate. Spectral imaging of synthetic plaques stained in the presence of live cells and lipid-binding protein albumin shows over 4000-fold Phazr-N₃ fluorescence intensity above background under no-wash conditions, and over 100-fold intensity above coplated microglial cells or a large excess of albumin. This use of a solvatochromic probe with structure-independent binding to free Aβ42 offers real-time, minimally invasive insight into the full Aβ42 aggregation pathway.

Protein oxidative ‘footprinting’— where obtaining structural information of proteins using hydroxyl radical labeling is detected with bottom-up proteomics mass spectrometry analysis—has evolved from an emerging technology to an approach applied to a range of structural biology and biophysics questions. Whether the hydroxyl radicals are generated from hydrogen peroxide (via photolysis, Fenton chemistry, or electrochemistry) or directly from water (through X-ray irradiation, plasma, or gamma rays), the resulting structural information is irreversibly encoded within the protein side chains and can be detected and quantitated with up to single-residue resolution, using a commonly applied set of LC-MS data reduction and analysis protocols. Quantitative measurements of changes in the oxidative labeling of amino acid side chain reflect alterations in solvent accessibility, typically resulting from protein-protein interactions, ligand binding, protein folding/conformational changes, or applied stress. Measuring changes in oxidative labeling thus provides a detailed map of structural changes and interaction sites across the system under study. Oxidative footprinting technologies over the past decade have demonstrated their utility as a mapping method for protein structure analysis in solution and within living cells. The goal of this article is to harmonize and disseminate best practices for experimental design, data collection, data interpretation, and data presentation. This perspective intends to facilitate the application of this insightful approach to new biological systems and experimental applications. The information presented in this article coalesces best practices independently developed by teams who have decades of experience in advancing protein oxidative footprinting. We provide a consensus viewpoint on the use of this technology and the different methodologies for structural elucidation in both academic research and in biopharmaceutical drug discovery and development.