Hydrogen Deuterium Exchange (HDX) for Protein Structure MS

Elucidating protein conformation, structure and dynamics with hydrogen deuterium exchange mass spectrometry

Utilizing a HDX-MS protocol takes advantage of the labile nature of protons present on protein backbone amides, and is a powerful tool in the study of protein structure. When dissolved in solution, proteins exchange these protons with hydrogen groups present in a deuterated buffer, and protons from the protein are exchanged with deuterium. Only the protons present on the backbone amides are measured. The rate of hydrogen to deuterium exchange provides solvent accessibility data, which can be used to infer information on protein structure and conformation. Mass spectrometry can be used to measure the rate of deuterium uptake.

HDX-MS analysis can be used to obtain information on structure, protein-protein or protein-ligand interaction sites, allosteric effects, intrinsic disorder, and conformational changes induced by posttranslational modifications (PTMs). HDX-MS has the advantage of not being limited by the size of proteins or protein complexes, and it is highly sensitive, able to detect coexisting protein conformations.


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How does HDX-MS work?

All HDX-MS experiments involve deuterium labeling prior to MS analysis. The protein is incubated in a deuterium buffer, which allows for the amide hydrogens present on the protein backbone to exchange with the deuterium buffer. The most commonly used labeling approach is continuous labeling, in which a protein in its steady state is incubated in deuterium buffer continuously over different time periods, and the exchange of hydrogen to deuterium is measured as a function of time. The time period can span from seconds to hours or days. After labeling, the samples are quenched by lowering the temperature of the experiment to 0°C and the pH of the reaction to 2.5. HDX-MS experiments can be performed in either a bottom-up or intact/top-down fashion.

HDX-MS workflows


The most commonly used strategy for HDX-MS is to digest the proteins into peptides and analyze them using mass spectrometry. This ensures complete sequence coverage and captures region-specific information from the protein. Before hydrogen-deuterium exchange is performed, the protein is digested and analyzed in a data-dependent fashion using multiple fragmentation techniques: collision-induced dissociation [CID], higher-energy collisional dissociation [HCD] and electron transfer dissociation [ETD]).

The goal is to identify as many overlapping peptides as possible. This is done to maximize sequence coverage of the protein for identification. This process is followed by the HDX-MS experiment. Since low pH is used in HDX-MS experiments to minimize deuterium back-exchange, acidic enzymes such as pepsin are preferred for digestion. The digestion can be performed in solution or on immobilized pepsin columns, the latter being the preferred approach.

Currently available commercial platforms, such as the H/D-X PAL Hydrogen Deuterium Exchange sampler system (LEAP Technologies), enable automated labeling and digestion. Upon digestion, the samples are desalted on a trap column and separated using reversed-phase chromatography prior to analysis by mass spectrometry.

Deuterium uptake information

The HDX-MS experiment can be performed in two ways. The first method is to collect full-scan MS to get deuterium uptake information on peptide levels to probe the protein conformation. The second method is to access higher resolution amino acid level information and thus requires acquiring peptide fragment level deuterium uptake data. Here a full-scan MS with data-dependent ETD MS2 is acquired for both unlabeled and deuterated samples. With this design, the full-scan MS experiment establishes the peptide-level deuterium incorporation value, while the higher resolution, single amino acid level deuterium incorporation value is obtained by ETD MS2. In this case, ETD is preferred over CID or HCD to avoid deuterium scrambling, a known phenomenon when energy-driven fragmentation methods are employed.

During scrambling, the protons on the peptide backbone migrate and don’t reflect the state of the peptides in solution. Studies have shown that ETD, a non-ergodic fragmentation technique, is far better suited as an activation choice due to the very low levels of hydrogen scrambling that occur during this process; therefore ETD allows for an accurate localization of incorporated deuterium at the single residue resolution level.

The alternative to bottom-up HDX-MS is intact/top-down analysis. In intact/top-down HDX-MS, proteins are introduced into the mass spectrometer after deuterium exchange without any digestion. For complex mixtures, some level of separation is performed before introducing proteins into the mass spectrometer, typically using a C4 column. Deuterium uptake may be measured at the intact level, or ETD may be employed to sequence the proteins.

Products for HDX-MS workflow

HDX MS Workflow
H/D-X PAL™ Hydrogen Deuterium Exchange sampler system (LEAP Technologies)

H/D-X PAL Hydrogen Deuterium Exchange sampler system (LEAP Technologies)

The H/D-X PAL autosampler with single head with syringe exchange has a standalone pepsin column chamber to enhance peptide coverage and a closed chamber for stable temperatures for all vials.  A flexible 3-valve configuration in the cooling chamber allows efficient sample cleanup. Chronos software provides full editing capabilities for method customization and full integration of Thermo Scientific Xcalibur software.

Acclaim 5μm PepMap 300 μ-Precolumns Cartridge Columns

Thermo Scientific Acclaim 5μm PepMap 300 μ-Precolumns Cartridge Columns are very short microcolumns consisting of a set of disposable cartridges, and they are currently available for purchase from

Generate high-resolution analyses of tryptic, natural, and synthetic peptides using Thermo Scientific Acclaim PepMap 100 C18 LC Columns. These columns are often used for LC-MS/MS peptide mapping for protein identification, biomarker discovery, and systems biology. Due to their high loading capacity, Acclaim PepMap 100 C18 columns are exceptionally suitable for the analysis of low abundant peptides in complex proteomics samples. Most of these C18 columns are pre-assembled with Thermo Scientific nanoViper Fingertight Fittings for easy installation. Classic columns are available as well.

Delivering the ultimate flexibility to expand experimental scope, and with built-in intelligence, Thermo Scientific Orbitrap Eclipse Tribrid Mass Spectrometer ensures the highest data quality for HDX-MS experiments. It delivers the high resolution-accurate mass necessary for specificity with short chromatographic runs required to prevent back exchange and to allow precise measurement of deuterium incorporation. Multiple fragmentation techniques, CID, HCD and ETD are available to identify as many overlapping peptides as possible, enabling maximum sequence coverage for protein identification. Plus, it offers ultimate precision with ETD fragmentation to allow localization of deuterium exchange to the amino acid level.

Types of information obtained using HDX-MS

A protein or protein complex can have multiple three-dimensional shapes, known as conformations. HDX-MS can provide information on the conformational differences between different states of a protein or protein complex, and can help elucidate a protein’s structured versus unstructured regions.

A protein or protein complex can undergo conformational changes to form new three-dimensional structures; this is known as protein dynamics. HDX-MS can provide information on the various short-lived, intermediate structures and the series of events that led from one state to another.

hdx-ms biomolecule binding

The process of biomolecule binding provides information on the interaction-interface between different subunits or ligands. The interactions may occur between a protein and another protein or between a protein and ligands such as nucleic acids, lipids, glycans and small molecules. The locations of the sites on the protein involved in the binding can be ascertained as well as how ligand binding affects protein conformation.

HDX-MS provides information on the effects of ligand binding on protein sites other than the binding site or throughout the whole protein.

The process provides information on proteins that lack a three-dimensional structure. This can be the entire protein or part of a protein that exists as flexible polypeptides or loops.

HDX-MS provides information on the regions involved in protein aggregation, conformational changes and the intermediate structures that form during aggregation.

HDX-MS literature integrated structural biology

SARS-CoV-2 Focus

  • A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection (Orbitrap Elite MS)
    Tingting Li, Hongmin Cai, Hebang Yao, Bingjie Zhou, Ning Zhang, Martje Fentener van Vlissingen, Thijs Kuiken, Wenyu Han, Corine H. GeurtsvanKessel, Yuhuan Gong, Yapei Zhao, Quan Shen, Wenming Qin, Xiao-Xu Tian, Chao Peng, Yanling Lai, Yanxing Wang, Cedric A. J. Hutter, Shu-Ming Kuo, Juan Bao, Caixuan Liu, Yifan Wang, Audrey S. Richard, Hervé Raoul, Jiaming Lan, Markus A. Seeger, Yao Cong, Barry Rockx, Gary Wong, Yuhai Bi, Dimitri Lavillette & Dianfan Li
    Nature Communications volume 12, Article number: 4635 (2021)
  • Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2 (Orbitrap Fusion Tribrid MS)
    Alexandra C. Walls, Brooke Fiala, Alexandra Schafer, Samuel Wrenn, Minh N. Pham, Michael Murphy, Longping V. Tse, Laila Shehata, Megan A. O’Connor, Chengbo Chen, Mary Jane Navarro, Marcos C. Miranda, Deleah Pettie, Rashmi Ravichandran, John C. Kraft, Cassandra Ogohara, Anne Palser, Sara Chalk, E-Chiang Lee, Kathryn Guerriero, Elizabeth Kepl, Cameron M. Chow, Claire Sydeman, Edgar A. Hodge, Brieann Brown, Jim T. Fuller, Kenneth H. Dinnon III, Lisa E. Gralinski, Sarah R. Leist, Kendra L. Gully, Thomas B. Lewis, Miklos Guttman, Helen Y. Chu, Kelly K. Lee, Deborah H. Fuller, Ralph S. Baric, Paul Kellam, Lauren Carter, Marion Pepper, Timothy P. Sheahan, David Veesler & Neil P. King
    Cell 183, 1367–1382, November 25, 2020

Ligand Binding

Technology Focus

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