Line G. Kristensen

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.

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.

X-ray footprinting mass spectrometry (XFMS) is a structural biology method that uses broadband X-rays for in situ hydroxyl radical labeling to map protein interactions and conformation in solution. However, while XFMS alone provides important structural information on biomolecules, as we move into the era of the interactome, hybrid methods are becoming increasingly necessary to gain a comprehensive understanding of protein complexes and interactions. Toward this end, we report the development of the first synergetic application of inline and real-time fluorescent spectroscopy at the Advanced Light Source's XFMS facility to study local protein interactions and global conformational changes simultaneously. To facilitate general use, we designed a flexible and optimum system for producing high-quality spectroscopy-XFMS hybrid data, with rapid interchangeable liquid jet or capillary sample delivery for multimodal inline spectroscopy, and several choices for optofluidic environments. To validate the hybrid system, we used the covalently interacting SpyCatcher–SpyTag split protein system. We show that our hybrid system can be used to detect the interaction of SpyTag and SpyCatcher via fluorescence resonance energy transfer (FRET), while elucidating key structural features throughout the complex at the residue level via XFMS. Our results highlight the usefulness of hybrid method in providing binding and structural details to precisely engineer protein interactions.

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 timepoints 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 24 h, and which culminate before the onset of any major fluorescence changes of conformation-specific aggregation fluorophore ThT. Solution anisotropy measurements show a low micromolar affinity of Phazr-N3 for non-aggregated Aβ42 in solution, and real-time measurements are consistent with rapid 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 shows over 4000-fold Phazr-N3 fluorescence intensity above background under no-wash conditions, and over 100-fold intensity above co-plated microglial cells. This use of solvatochromic probes and kinetic anisotropy offers real-time, minimally invasive insight into Aβ42 aggregation and the molecular mechanisms of neurodegeneration.

X-ray footprinting coupled with mass spectrometry (XFMS) presents a novel approach in structural biology, offering insights into protein conformation and dynamics in the solution state. The interaction of the cancer-immunotherapy monoclonal antibody nivolumab with its antigen target PD-1 was used to showcase the utility of XFMS against the previously published crystal structure of the complex. Changes in side-chain solvent accessibility, as determined by the oxidative footprint of free PD-1 versus PD-1 bound to nivolumab, agree with the binding interface side-chain interactions reported from the crystal structure of the complex. The N-linked glycosylation sites of PD-1 were confirmed through an LC-MS/MS-based deglycosylation analysis of asparagine deamidation. In addition, subtle changes in side-chain solvent accessibility were observed in the C′D loop region of PD-1 upon complex formation with nivolumab.

In the method of X-ray footprinting mass spectrometry (XFMS), proteins at micromolar concentration in solution are irradiated with a broadband X-ray source, and the resulting hydroxyl radical modifications are characterized using liquid chromatography mass spectrometry to determine sites of solvent accessibility. These data are used to infer structural changes in proteins upon interaction with other proteins, folding, or ligand binding. XFMS is typically performed under aerobic conditions; dissolved molecular oxygen in solution is necessary in many, if not all, the hydroxyl radical modifications that are generally reported. In this study we investigated the result of X-ray induced modifications to three different proteins under aerobic versus low oxygen conditions, and correlated the extent of damage with dose calculations. We observed a concentration-dependent protecting effect at higher protein concentration for a given X-ray dose. For the typical doses used in XFMS experiments there was minimal X-ray induced aggregation and fragmentation, but for higher doses we observed formation of covalent higher molecular weight oligomers, as well as fragmentation, which was affected by the amount of dissolved oxygen in solution. The higher molecular weight products in the form of dimers, trimers, and tetramers were present in all sample preparations, and, upon X-ray irradiation, these oligomers became non-reducible as seen in SDS-PAGE. The results provide an important contribution to the large body of X-ray radiation damage literature in structural biology research, and will specifically help inform the future planning of XFMS, and well as X-ray crystallography and small-angle X-ray scattering experiments.