Hydroxyl Radical Protein Footprinting: A Mass Spectrometry-Based Structural Method for Studying the Higher Order Structure of Proteins

Abstract

Hydroxyl radical protein footprinting (HRPF) coupled to mass spectrometry has been successfully used to investigate a plethora of protein-related questions. The method, which utilizes hydroxyl radicals to oxidatively modify solvent-accessible amino acids, can inform on protein interaction sites and regions of conformational change. Hydroxyl radical-based footprinting was originally developed to study nucleic acids, but coupling the method with mass spectrometry has enabled the study of proteins. The method has undergone several advancements since its inception that have increased its utility for more varied applications such as protein folding and the study of biotherapeutics. In addition, recent innovations have led to the study of increasingly complex systems including cell lysates and intact cells. Technological advances have also increased throughput and allowed for better control of experimental conditions. In this review, we provide a brief history of the field of HRPF and detail recent innovations and applications in the field.

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

“…817,821,822 This technique generates chemical reactions on the microsecond timescale, which can reveal the solvent-accessible residues of thousands of proteins from about 10 million cells, [823][824][825][826] or from 10,000 Caenorhabditis elegans. 827,828 While FPOP in vitro has been shown to be capable of characterizing epitope mapping, conformational changes and even of yielding good protein structure predictions, 821,829 it is still in a development phase in cells. 830,831 Other MS proteomics strategies have been designed to map the binding-proteome of small molecules that cross-link either with reactive residues of proteins (cysteines, lysines) or upon photo-activation.…”

Magnetoelectric dissociation of Alzheimer’s β-amyloid aggregates

“…According to the literature ( 41 – 43 ), free radical species can posttranslationally modify the amino acid sequence of Aβ fibrils by depriving electrons from Aβ peptides to induce structural instability. For example, histidine (His 6 ) in Aβ peptides can be converted to 2-oxo-histidine by the action of •OH and •O 2 − (fig.…”

A proximity labeling method for protein–protein interactions on cell membrane

“…Hydroxyl radical protein footprinting (HRPF) is commonly used for the determination of protein structure and protein-ligand interactions, where the interaction sites are characterized by the site-specic oxidation of proteins and mass spectrometry. 20,21 Combining proximity labeling and hydroxyl radical protein footprinting, Li and coworkers developed a labeling approach by immobilizing a catalytic Fe(III) probe on cell membrane sialic acids to map the proteins interacting with sialylated glycans through site-specic oxidation. 22 The enrichment of interacting proteins was not required with this approach, which assess the extent of the proteinprotein interactions by quantifying the degree of oxidation at interaction sites.…”