Hydrogen deuterium exchange (HDX) mass spectrometry (MS) is a powerful tool for studying the dynamics of higher order structure of protein-based therapeutics. The rate of hydrogen-to-deuterium exchange within the amide hydrogen on the backbone of biotherapeutics provides solvent accessibility information, and thus protein structure and conformation can be inferred.

Thermo Scientific Orbitrap-based HDX MS offers a robust method for the analysis of protein conformation, conformation dynamics, and protein-protein interactions.

Full structural characterization is critical in biopharmaceutical development where the subtle but critical local conformational changes can impact safety and efficacy.

Thermo Scientific workflow for hydrogen deuterium exchange mass spectrometry

Hydrogen deuterium exchange (HDX) workflow
Any Orbitrap-based system can be coupled to the H/D-X PAL/LC front end

Bottom-up HDX-MS analysis

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].

Currently available commercial platforms, such as the H/D-X PAL Hydrogen Deuterium Exchange sampler system (LEAP Technologies), enable automated labelling and digestion. 

Intact/top-down HDX-MS analysis

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.

H/D-X PAL Hydrogen Deuterium Exchange Sampler system

H/D-X PAL Hydrogen Deuterium Exchange Sampler system

LEAP Technologies

The new LEAP HDX extended parallel system can accurately schedule fast deuterium exchange time points (approximately 15 seconds) to probe protein conformation dynamics. The H/D-X PAL autosampler includes: 

  • A standalone enzyme column
  • A chamber to perform on-line peptide digestion
  • A closed chamber for stable temperatures for all vials

A flexible 3-valve configuration in the cooling chamber allows efficient sample clean-up. The updated three valve system could set up the back-flash function to efficiently reduce the carryover and clean up the build-up on top of the column which results in very reproducible chromatography. Chronos software provides full editing capabilities for method customization and full integration of Thermo Scientific Xcalibur software.

HPLC Columns

Trap column

Trap column

Generate high-resolution analyses of tryptic, natural, and synthetic peptides using Acclaim PepMap HPLC columns. Due to their high loading capacity, these columns are exceptionally suitable for the analysis of low abundant peptides in complex samples.

Analytical column

Analytical column

Achieve exceptional peak shape and resolution for your LC/MS applications with Hypersil GOLD HPLC columns. These endcapped, ultrapure, silica-based columns deliver significant reduction in peak tailing using generic gradients with C18 selectivity. With their excellent resolution, efficiency, and sensitivity, Hypersil GOLD columns give you confidence in the accuracy and quality of your analytical data.

HPLC system

Vanquish Neo UHPLC system

Pursue the next scientific breakthrough with the best performance, productivity, and usability in a nano-, capillary-, and micro-flow UHPLC systems. The Vanquish Neo UHPLC system combines an unrivaled degree of innovation to deliver 24/7 reproducible separations of complex mixtures at maximum performance for a variety of high-sensitivity LC-MS workflow.

Achieving next-level performance for HDX-MS with the Thermo Scientific Orbitrap Exploris 240 mass spectrometer

Orbitrap Eclipse Tribrid Mass Spectrometer

Delivering the ultimate flexibility to expand experimental scope, and with built-in intelligence, 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, UVPD 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 or UVPD fragmentation to allow localization of deuterium exchange to the amino acid level.

Learn more ›


Featured webinars

Running the Gamut - Tales of Protein Dynamics in Drug Discovery,

Benjamin T. Walters. Scientist, Genentech

View webinar

Application of HDX-MS for Drug Discovery & Development
Pfizer & BioAnalytix: Improved Biotherapeutic Characterization using HDX-M

No records were found matching your criteria

JournalTitleAuthorsFeatured MS System
Nature Communications volume 12, Article number: 2547 (2021)Selection of a picomolar antibody that targets CXCR2-mediated neutrophil activation and alleviates EAE symptoms  Xiaojie Shi, et al.Orbitrap Fusion
Nature Communications volume 12, Article number: 4635 (2021)   A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection Tingting Li, et al. Orbitrap Elite
Cell 183, 1367–1382, November 25, 2020 Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2   Alexandra C. Walls, et al.Orbitrap Fusion
Nature Communications volume 12, Article number: 1538 (2021)  Naturally acquired blocking human monoclonal antibodies to Plasmodium vivax reticulocyte binding protein 2b Li-Jin Chan, et al.Orbitrap Velos Pro  
Nature Microbiology volume 6, pages 921–931 (2021)   Cryo-EM structures of Lassa and Machupo virus polymerases complexed with cognate regulatory Z proteins identify targets for antivirals complementary with cryo EM Xin Xu, et al.Q Exactive   
Nature Communications volume 11, Article number: 6435 (2020)  Bivalent antibody pliers inhibit β-tryptase by an allosteric mechanism dependent on the IgG hinge Henry R. Maun, et al.Orbitrap Elite  
Nature Communications volume 10, Article number: 1320 (2019)   A post-translational modification of human Norovirus capsid protein attenuates glycan binding Alvaro Mallagaray, et al.Orbitrap Fusion
Structure 26, 1651–1663, December 4, 2018  Hydrogen-Deuterium Exchange Coupled to Top and Middle-Down Mass Spectrometry Reveals Histone Tail Dynamics before and after Nucleosome Assembly  Kelly R. Karch, et al.Orbitrap Fusion
Nature volume 546, pages 259–264 (2017) Structure of the full-length glucagon class B G-protein-coupled receptor   Haonan Zhang, et al.Q Exactive
Structure 26, 1651–1663, December 4, 2018  Hydrogen-Deuterium Exchange Coupled to Top and Middle-Down Mass Spectrometry Reveals Histone Tail Dynamics before and after Nucleosome Assembly  Kelly R., et al.Orbitrap Fusion
Analytica Chimica Acta Volume 1143, 25 January 2021, Pages 65-72   High-throughput hydrogen deuterium exchange mass spectrometry (HDX-MS) coupled with subzero-temperature ultrahigh pressure liquid chromatography (UPLC) separation for complex sample analysis   Mulin Fang et al.Orbitrap Elite  
Methods Volume 184, 1 December 2020, Pages 135-140 Dual protease type XIII/pepsin digestion offers superior resolution and overlap for the analysis of histone tails by HX-MS   James Mullahoo, et al.Orbitrap Fusion Lumos
Analytical Chemistry  2018, 90, 5, 3079-3082  Top-Down Hydrogen–Deuterium Exchange Analysis of Protein Structures Using Ultraviolet Photodissociation  Nicholas I. Brodie, et al.Orbitrap Fusion Lumos
Analytical Chemistry 2018, 90, 11, 6409-6412  Automated Removal of Phospholipids from Membrane Proteins for H/D Exchange Mass Spectrometry Workflows  Kyle W. Anderson, et al.Orbitrap Elite MS
Thermo Scientific Publication List (2014 to 2017) Key HDX-MS Publications for Pharma & BioPharma  MultipleMultiple

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