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Mass spectrometry-based hydrogen-deuterium exchange analysis
Proteins are not static, they breath, shift and reorganize as they bind partners, respond to their environment, and perform their biological roles. To help you pinpoint where interactions reshape the protein’s dynamic landscape, hydrogen-deuterium exchange mass spectrometry (HDX-MS) captures this motion by tracking how backbone amide hydrogens exchange with deuterium over time. The resulting exchange patterns provide a sensitive readout of structural flexibility, solvent accessibility, conformational stability and allosteric changes.
To quantify deuterium uptake kinetics at the peptide or residue levels, HDX-MS uses time-resolved D2O labeling with rapid quench conditions, controlled proteolysis, and LC-MS analysis. These uptake profiles are then processed through analysis to generate differential HDX maps that localize binding interfaces and conformational changes across proteins and complexes. Thermo Scientific mass spectrometers deliver the sensitivity, resolution and reproducibility needed for confident peptide identification and robust deuterium quantification, supporting high-coverage, scalable HDX-MS studies from sample preparation through data interpretation.
Benefits of the HDX-MS platform
Fully automated, end-to-end workflow
For streamlined acquisition and method control, integrated HDX automation (Trajan CHRONECT platform) is controlled by CHRONOS and fully integrated with Thermo Scientific Xcalibur Software.
Flexible UHPLC option
Experience nano-/capillary-/micro-flow all-in-one; robust operation up to 1500 bar and biocompatible high-pressure gradient pump for sample loading with Thermo Scientific Vanquish Binary Pump N and Vanquish Flex pump.
Orbitrap performance
High-resolution, accurate-mass Orbitrap detection supports the specificity needed for short HDX-MS runs and enables precise measurement of deuterium incorporation.
Higher-resolution Localization
Support more localized structural information with ETD and UVPD fragmentation.
Advanced capabilities of HDX-MS platform |
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Library based (DDA) MS1 mode |
High resolution and deep coverage |
Library based DIA mode (HCD based) |
Automated data analysis and high throughput |
Intact level HDX-MS |
Fast screening and condition optimization |
Bottom-up and Top-down ETD/UVPD |
Improved resolution down to residue level |
Low-flow HDX-MS (cap/nano-flow) |
Boost sensitivity and low sample consumption |
Thermo Scientific HDX-MS workflow
Measure protein conformational dynamics and interaction-driven structural changes with Thermo Scientific Orbitrap mass spectrometer-based HDX-MS workflow, which delivers a streamlined, end-to-end solution. The workflow combines automated D2O labeling, quench, and enzymatic digestion on the CHRONET HDX platform with highly reproducible Thermo Scientific Vanquish UHPLC System separations. High-resolution, accurate-mass Orbitrap acquisition provides the spectral quality needed for confident peptide identification and precise measurement of deuterium incorporation across timepoints.
Flexible data analysis options, including Thermo Scientific Proteome Discoverer Software and Thermo Scientific BioPharma Finder Software, together with HDExaminer and HDExaminer Pro, enable efficient generation of uptake curves and differential HDX maps form sample preparation through data interpretation.
Low-flow HDX-MS workflow
HDX-MS is an established technique for probing protein structure and dynamics. However, its application to large, heterogeneous, or sample-limited systems can be constrained by sensitivity and material requirements associated with conventional micro-flow workflows, which can hinder the study of biologically relevant targets such as large protein assemblies, membrane proteins, and low-abundance complexes.
Orbitrap mass spectrometers and Vanquish binary pump N based capillary-flow HDX-MS workflow can deliver a marked improvement in analytical sensitivity while maintaining high robustness and throughput on a fully automated HDX-MS platform.
Low–flow HDX-MS
What is possible with traditional HDX-MS |
What we offer |
Microflow – 50µL/min |
Capillary flow – 5 µL/min |
Analytical flow – >200 µL/min |
Nanoflow – <1nL/min |
What are the benefits?
- Increased complexity of samples
- Decreased consumption of samples
DIA-HDX-MS workflow
Data-independent acquisition HDX-MS (DIA-HDX-MS), a next-generation workflow, enhances traditional HDX-MS by collecting both precursor and fragment ion data simultaneously, enabling automated and highly reliable peptide identification and deuterium uptake analysis. By leveraging DIA acquisition, the workflow increases redundancy and confidence, accelerates processing, and extends the applicability of HDX-MS to complex and high-throughput samples.
Thermo Scientific mass spectrometers with high-resolution and accurate-mass detection combined with HDExaminer Pro software delivers deeper structural insights into protein conformational dynamics and interactions with improved reproducibility and throughout.
What are the benefits?
- Pain point: manual assessment and validation of spectral data.
- High-throughput: week of manual validation vs. auto-validation
Single-residue HDX-MS workflow
For residue-level resolution of conformational dynamics and interaction interfaces, single-residue HDX-MS extends traditional peptide-level HDX by combining solution-phase deuterium labeling with gas-phase fragmentation strategies that preserve deuterium localization. Thermo Scientific HDX-MS platform uniquely supports this approach through multiple fragmentation techniques, including ETD, EThcD, and UVPD. These electron-based and photodissociation methods minimize hydrogen scrambling compared to purely collisional approaches enabling more accurate residue-specific mapping.
Thermo Scientific Orbitrap Tribrid mass spectrometers incorporate special calibration and tuning strategies designed to operate under low-scrambling conditions, helping preserve solution-phase labeling patterns during gas-phase activation. Together, high-resolution, accurate-mass detection and multi-fragmentation flexibility provide a powerful solution for high-confidence, single-residue HDX-MS analysis of proteins and complexes.
Single-residue HDX-MS
What is currently available |
What we offer |
Bottom-up HCD |
Bottom-up ETD/Top-down |
Peptide level resolution |
Residue level resolution |
What are the benefits?
- Improved resolution: Peptide level to residue level
Single-residue HDX resolution
Thermo Scientific Orbitrap Ascend Structural Biology Mass Spectrometer, featuring ETD and EThcD fragmentation, delivers single-residue HDX resolution supported by specialized low-scrambling calibration and optimized activation conditions.
What scientists are saying about Orbitrap Excedion Pro mass spectrometer
“The Orbitrap Exploris 480 mass spectrometer has been essential for our high-throughput HDX-MS workflows, consistently delivering reproducible and reliable peptide-level deuterium uptake data. The Orbitrap Excedion Pro mass spectrometer elevates this capability through rapid ETD fragmentation.
Our initial evaluation demonstrates high-quality single-residue resolution data with minimal deuterium scrambling, enabling precise identification of specific residues involved in protein-ligand and protein-protein interactions—a significant advance beyond traditional peptide-level analysis. These capabilities will facilitate our understanding of binding interfaces, establishing Orbitrap Excedion Pro mass spectrometer as an invaluable platform for next-generation HDX-MS studies.”
Malvina Papanastasiou, PhD
Group Leader, Research Scientist
Broad Institute of MIT & Harvard
For Research Use Only. Not for use in diagnostic procedures.