Abstract
Protein footprinting mediated by mass spectrometry has evolved over the last 30 years from proof of concept to commonplace biophysics tool, with unique capabilities for assessing structure and dynamics of purified proteins in physiological states in solution. This review outlines the history and current capabilities of two major methods of protein footprinting: reversible hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting (HRF), an irreversible covalent labeling approach. Technological advances in both approaches now permit high-resolution assessments of protein structure including secondary and tertiary structure stability mediated by backbone interactions (measured via HDX) and solvent accessibility of side chains (measured via HRF). Applications across many academic fields and in biotechnology drug development are illustrated including: detection of protein interfaces, identification of ligand/drug binding sites, and monitoring dynamics of protein conformational changes along with future prospects for advancement of protein footprinting in structural biology and biophysics research.
Highlights
TO FOOTPRINTING: ROOTS AND HISTORY “Footprinting” based technologies were initially developed to understand nucleic acids structure and dynamics
We introduce a figure of merit for comparing structural resolution across protein footprinting experiments defined as the number of probe sites identified and quantified divided by the total number of residues, expressed as a %
We examined over 60 manuscripts reporting 75 footprinting results primarily from the last 10 years to understand the evolving approaches across many laboratories. supplemental Table S1 summarizes the survey results, including 38 hydrogen-deuterium exchange (HDX)-MS results and 31 hydroxyl radical footprinting (HRF)-MS results (16 using laser photolysis of peroxide and 15 using radiolysis), and seven other covalent labeling-MS results (1 acetylation, 3 GEE and 3 carbene labeling)
Summary
TO FOOTPRINTING: ROOTS AND HISTORY “Footprinting” based technologies were initially developed to understand nucleic acids structure and dynamics. Studies included hydrogen exchange footprinting to examine protein structure (advanced by Rosa and Richards) [7, 8] whereas alternative approaches to probe conformational change used enzymatic cleavage and gel analysis (e.g. similar to those of nucleic acids footprinting) to successfully define Ab epitopes [9]. Footprinting experiments typically need to balance the need for comprehensive labeling (required to get a good signal across many sites) while avoiding artifacts of the labeling process itself In this respect HDX-MS is advantageous as it represents a minimal perturbation of the protein. For other protein labeling methods, which are typically irreversible, a detailed understanding of the chemistry of the individual reagents, development of appropriate dosimetry measurements, and developing quantitative mass spectrometry readouts of modified species with adequate dynamic range have been critical to progress. Description of Labeling Technologies for Footprinting: Deuterium and OH Radical Labeling Probes Backbone and Side-
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