Future directions of structural mass spectrometry using hydroxyl radical footprinting
- 1 September 2010
- journal article
- review article
- Published by Wiley in Journal of Mass Spectrometry
- Vol. 45 (12), 1373-1382
- https://doi.org/10.1002/jms.1808
Abstract
Hydroxyl radical protein footprinting coupled to mass spectrometry has been developed over the last decade and has matured to a powerful method for analyzing protein structure and dynamics. It has been successfully applied in the analysis of protein structure, protein folding, protein dynamics, and protein–protein and protein–DNA interactions. Using synchrotron radiolysis, exposure of proteins to a ‘white’ X‐ray beam for milliseconds provides sufficient oxidative modification to surface amino acid side chains, which can be easily detected and quantified by mass spectrometry. Thus, conformational changes in proteins or protein complexes can be examined using a time‐resolved approach, which would be a valuable method for the study of macromolecular dynamics. In this review, we describe a new application of hydroxyl radical protein footprinting to probe the time evolution of the calcium‐dependent conformational changes of gelsolin on the millisecond timescale. The data suggest a cooperative transition as multiple sites in different molecular subdomains have similar rates of conformational change. These findings demonstrate that time‐resolved protein footprinting is suitable for studies of protein dynamics that occur over periods ranging from milliseconds to seconds. In this review, we also show how the structural resolution and sensitivity of the technology can be improved as well. The hydroxyl radical varies in its reactivity to different side chains by over two orders of magnitude, thus oxidation of amino acid side chains of lower reactivity are more rarely observed in such experiments. Here we demonstrate that the selected reaction monitoring (SRM)‐based method can be utilized for quantification of oxidized species, improving the signal‐to‐noise ratio. This expansion of the set of oxidized residues of lower reactivity will improve the overall structural resolution of the technique. This approach is also suggested as a basis for developing hypothesis‐driven structural mass spectrometry experiments. Copyright © 2010 John Wiley & Sons, Ltd.Keywords
This publication has 54 references indexed in Scilit:
- Time-Dependent Changes in Side-Chain Solvent Accessibility during Cytochrome c Folding Probed by Pulsed Oxidative Labeling and Mass SpectrometryJournal of Molecular Biology, 2010
- Integrated Algorithms for High-Throughput Examination of Covalently Labeled Biomolecules by Structural Mass SpectrometryAnalytical Chemistry, 2009
- Probing protein structure by amino acid‐specific covalent labeling and mass spectrometryMass Spectrometry Reviews, 2008
- Concurrent nucleation of 16S folding and induced fit in 30S ribosome assemblyNature, 2008
- Synchrotron Protein Footprinting Supports Substrate Translocation by ClpA via ATP-Induced Movements of the D2 LoopStructure, 2008
- Analysis of the Oxidative Damage-Induced Conformational Changes of Apo- and Holocalmodulin by Dose-Dependent Protein Oxidative Surface MappingBiophysical Journal, 2007
- Visualizing Arp2/3 complex activation mediated by binding of ATP and WASp using structural mass spectrometryProceedings of the National Academy of Sciences of the United States of America, 2007
- Fast Fenton footprinting: a laboratory-based method for the time-resolved analysis of DNA, RNA and proteinsNucleic Acids Research, 2006
- Unfolding of Apomyoglobin Examined by Synchrotron FootprintingBiochemical and Biophysical Research Communications, 2001
- Radiolytic Protein Surface MappingBiochemical and Biophysical Research Communications, 1996