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Mapping Protein-Ligand Interactions with Proteolytic-Digestion, Hydrogen/Deuterium Exchange-Mass Spectrometry



Jeffrey W. Hudgens, Elyssia S. Gallagher


range from small organic and inorganic molecules to lipids, nucleic acids, peptides, and proteins. Along the protein chain making up such proteins, resident amide groups constantly exchange protons with water. When immersed in heavy water, mass spectrometry (MS) can measure the change of mass associated with the hydrogen-deuterium exchange (HDX). Protein-ligand interactions reduce or enhance the hydrogen exchange rates of the amide protons, and the measurement of the amide exchange rates can provide rich information regarding the dynamical structure of the protein-ligand complex. This paper describes a protocol for conducting bottomup, continuous uptake, proteolytic fragmentation HDX-MS experiments that can help identify and map the interacting peptides of a protein-ligand interaction. This tutorial outlines the fundamental theory governing hydrogen exchange, provides practical information regarding the preparation of protein samples and the preparation of solutions used to conduct the exchange reaction, reaction quenching, enzymatic digestion, and the chromatographic separation of peptides prior to measurement by a mass spectrometer. Tables list representative combinations of fluidic components used by HDX-MS researchers and summarize the available HDX-MS analysis software packages. Methods for achieving unit amide resolution with electron transfer dissociation (ETD) and electron capture dissociation are discussed. Two HDX-MS case studies describe the determination of protein-ligand interactions involving: 1) a continuous sequence of interacting residues and 2) a set of discontinuously numbered residues, residing spatially near each other that interact with an antigenic epitope.
Methods In Enzymology


Hydrogen/Deuterium Exchange, Mass Spectrometry, Hydrogen/Deuterium Exchange-Mass Spectrometry, protein, Protein-Ligand complex, Protein-Ligand Interaction


Hudgens, J. and Gallagher, E. (2015), Mapping Protein-Ligand Interactions with Proteolytic-Digestion, Hydrogen/Deuterium Exchange-Mass Spectrometry, Methods In Enzymology, [online], (Accessed June 13, 2024)


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Created October 23, 2015, Updated November 10, 2018