Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) Service

Creative Proteomics provides end-to-end HDX-MS analysis to reveal protein higher-order structure (HOS), binding interfaces, and stability—helping researchers and biopharma teams make confident, data-driven decisions.

Comprehensive Coverage — Up to 95% sequence coverage with near-residue resolution
Validated for Challenging Targets — Antibodies, ADCs, complexes, and membrane proteins
Quantitative Confidence — Replicate precision ≤10% with robust statistical testing
Decision-Ready Results — Binding sites, conformational changes, comparability reports

From antibody epitope mapping to formulation stability assessment, our HDX-MS service delivers structural clarity and reliable data for biologics research.

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What Is Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS)?

Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) is a high-resolution method to study protein higher-order structure and dynamics. By measuring how backbone amide hydrogens in a protein exchange with deuterium in D₂O, HDX-MS provides direct insights into structural stability, solvent accessibility, and conformational flexibility. Regions that are exposed or dynamic show rapid exchange, while folded or protected domains exchange more slowly. After controlled labeling, samples are quenched, digested, separated at low temperature, and analyzed by high-resolution LC-MS.

The resulting data reveal binding interfaces, epitope footprints, allosteric effects, and comparability across different protein states, making HDX-MS a critical tool for protein characterization, antibody mapping, biosimilar assessment, and formulation studies.

Scientific Questions Addressed by HDX-MS

Where does a ligand bind? Map epitope/paratope and small-molecule binding sites; rank binding hotspots by protection magnitude.
Does my protein change conformation upon binding or stress? Localize stabilized/destabilized segments, hinge motions, and allosteric pathways.
Are biosimilar or variant molecules conformationally comparable? Quantify similarity at the HOS level beyond intact mass and peptide mapping.
Which formulation best preserves native dynamics? Compare buffers, excipients, pH, and temperature histories for HOS stability.
How do PTMs or mutations affect local dynamics? Resolve protection/exposure linked to sequence changes.
Do assembly states or protein–protein interactions alter solvent accessibility? Reveal oligomer interfaces and complex formation.
What is the mechanism of action at the domain or loop level? Connect functional states to dynamic fingerprints.

Advantages of Our HDX-MS Service

Tight Temperature & pH Control — Reliable Exchange Measurements

On-line quench/digestion and sub-ambient chromatography (≈0–4 °C) minimize back-exchange. Typical deuterium recovery ≥70% ensures accurate kinetic profiling from seconds to hours.

High Sequence Coverage — Up to 85–95% for Soluble Proteins

Optimized digestion with immobilized pepsin and auxiliary acid proteases frequently delivers >90% sequence coverage, with overlapping peptides providing ~5–10 residue spatial resolution.

Residue-Level Resolution — Near-Residue Insights with ETD/ECD

Extensive overlapping peptide coverage combined with ETD/ECD fragmentation narrows protection mapping to near-residue detail while minimizing hydrogen scrambling.

Quantitative Rigor — Statistical Confidence for Every Dataset

Biological and technical replicates typically achieve RSD ≤10%. Uptake differences are evaluated with per-peptide significance testing (Welch's t-test, Benjamini–Hochberg adjustments), with global alignment and drift correction.

Robust for Challenging Targets — Validated on Complex Proteins

Workflows are compatible with antibodies, large complexes, disordered proteins, glycoproteins, and membrane proteins, supported by detergent/lipid/amphipol screening.

Orthogonal Enhancements — More Than HDX-MS Alone

Optional ion mobility separates conformers, native MS provides intact-level monitoring, and back-exchange benchmarks are included to validate data reliability.

Reproducibility & Precision — Consistency Across Studies

Peptide-level replicate precision commonly ≤10% variation. QC gates ensure that acceptance thresholds for coverage, recovery, and reproducibility are met before reporting.

Technical Services

Service Scope Workflow and Instrumentation Application Sample Requirement Deliverables Case FAQ Get a Custom Proposal

Scope of Our HDX-MS Service Solutions

Epitope Mapping and Paratope Mapping

Define antibody–antigen interaction sites with peptide- to residue-level resolution, enabling clone selection, affinity ranking, and mechanism-of-action studies.

Ligand Binding & Small Molecule Footprinting

Identify and compare binding sites for small molecules, fragments, nucleic acids, and cofactors to guide structure–activity relationship (SAR) studies.

Comparability & Higher-Order Structure (HOS) Assessment

Evaluate biosimilars, variants, and production lots for conformational similarity, supporting quality control, regulatory filings, and process consistency.

Formulation & Stability Studies

Screen buffers, excipients, and storage conditions to determine which best preserve native protein folding and dynamics under stress or long-term conditions.

Allostery & Conformational Dynamics

Detect subtle structural rearrangements and long-range communication pathways upon ligand binding, activation, or inhibition.

Protein–Protein & Complex Assembly Mapping

Characterize oligomerization interfaces, multi-subunit assemblies, and dynamic association/dissociation behaviors.

Membrane Protein & Challenging Targets

Specialized workflows with detergent, lipid, and amphipol systems ensure compatibility and structural insights for receptors, transporters, and glycoproteins.

Mutation & Post-Translational Modification (PTM) Effects

Localize dynamic changes caused by sequence variants or PTMs, providing mechanistic understanding of functional alterations.

Analyte Types Supported by HDX-MS Service:

Monoclonal and Bispecific Antibodies: High coverage mapping of Fab and Fc regions, suitable for epitope/paratope studies and comparability assessments.
Antibody–Drug Conjugates (ADCs): Evaluation of conjugation-induced structural changes and stability under formulation or stress conditions.
Protein Complexes and Multisubunit Assemblies: Characterization of oligomerization interfaces, subunit dynamics, and complex formation.
Membrane Proteins and Transporters: Specialized workflows with detergent, lipid, or amphipol systems to preserve native conformations.
Enzymes and Signaling Proteins: Analysis of active/inactive states, allosteric transitions, and conformational rearrangements.
Nucleic Acid–Protein Complexes: Mapping of DNA/RNA binding interfaces and conformational effects of nucleic acid association.
Glycoproteins and Post-Translationally Modified Proteins: Assessing the impact of glycosylation, phosphorylation, and other PTMs on local structural stability.
Peptides and Small Proteins: High-sensitivity workflows designed for limited quantities or intrinsically disordered proteins.

HDX-MS Analysis Workflow at Creative Proteomics

Consultation & Experimental Design

Define the biological question, sample states (apo/bound, formulations), labeling windows (seconds to hours), replicates, and statistical thresholds. Select compatible additives/detergents if needed.

Sample Qualification

Intact mass and preliminary peptide map under quench conditions; rapid compatibility screen for buffers, salts, and excipients that might impair labeling or chromatography.

Deuterium Labeling

Controlled D₂O exposure across multiple timepoints to capture exchange kinetics; on-ice handling and precise timing to ensure consistency.

Quench & On-line Proteolysis

Low pH quench, sub-ambient on-line pepsin (and auxiliary acid proteases) digestion to produce robust overlapping peptides.

Cold LC-MS Acquisition

Sub-ambient UPLC separates deuterated peptides; high-resolution MS (Orbitrap and/or Q-TOF) acquires isotopic envelopes with low back-exchange.

Data Processing & Statistics

Automated peptide identification, deuterium uptake calculation, time-course fitting, replicate statistics, significance mapping, and structural projection when PDB/AlphaFold models are provided.

Interpretation & Reporting

Clear, decision-ready deliverables: uptake curves, differential maps, per-peptide statistics, and an executive narrative linked to your study goals.

HDX-MS Instrumentation and Platform Capabilities

Automated HDX Platform — Temperature-controlled autosampler (≈0–4 °C) with on-line labeling, quench, and digestion; independent immobilized pepsin column, compatible with auxiliary acid proteases.

Sub-Ambient UPLC — Trap-and-elute separation with low-dead-volume plumbing and cold flow paths; C18 columns for peptide-level, C4 for intact proteins.

High-Resolution MS — Orbitrap (up to ~240k resolution, <2 ppm accuracy) and Q-TOF (fast, broad dynamic range); optional ETD/ECD, CID/HCD, ion mobility, and native MS.

QC Hardware — Lock-mass infusion, column switching, blank/decontamination channels to ensure robust and reproducible performance.

Thermo Scientific Vanquish Flex UHPLC

Orbitrap Exploris 480/240

Q Exactive HF-X

Applications of HDX-MS Service

Biopharmaceutical Development

Assess higher-order structure comparability, monitor formulation effects, and support biosimilar development.

Antibody & Biologics

Characterize Fc or glyco-engineering, bispecifics, and antibody–drug conjugates (ADC).

Vaccine & Antigen Design

Reveal epitope exposure, conformational states, and immune-relevant dynamics.

Enzyme Mechanism

Capture allosteric regulation, cofactor binding, and transient catalytic intermediates.

Protein Engineering

Evaluate mutation impacts, folding stability, and synthetic scaffold designs.

Membrane Proteins

Study transporters, channels, and receptors in lipid or detergent systems.

Sample Requirements for HDX-MS Projects

Item	Requirement (Typical)	Notes
Sample type	Purified protein/antibody/antigen or defined complex	Monodisperse preferred
Purity	≥90% (LC-MS or SDS-PAGE)	Lower purity possible after consultation
Concentration	0.5–20 mg/mL working range	Adjusts with MW/ionization efficiency
Volume per condition	≥50–200 μL	Scales with timepoints and replicates
Buffer (exchange state)	HDX-compatible, low-amine	Recommend phosphate/acetate/formate; avoid Tris/glycine
Detergents/lipids	HDX-qualified only	e.g., DDM, LMNG, CHAPS, amphipols; pre-screen recommended
Quench compatibility	Tolerates rapid drop to pH ≈ 2.5 and low temperature	No precipitation/phase separation
Stability	Stable on ice; minimize freeze–thaw	Non-amine stabilizers if needed
Sequence/structure	FASTA required; PDB/AlphaFold optional	Enables coverage planning & 3D mapping
Ligands/controls (optional)	Provide antigen/small molecule/cofactor with known stoichiometry	Supports epitope/binding studies
Shipping & storage	Cold chain (ice packs or dry ice as appropriate)	Include lot ID and handling notes

Deliverables: What You Get from Our HDX-MS Service

Final Report — Summary of study design, key findings (binding sites, conformational changes, comparability), and conclusions.
Raw Data — Vendor MS raw files (Orbitrap/Q-TOF) and LC-MS method settings.
Processed Data Tables — Peptide coverage, deuterium uptake values, differential ΔD statistics.
Visualizations — Uptake curves, isotopic envelopes, Woods/butterfly plots, sequence heat maps, optional 3D structural mapping.
Quality Metrics — Sequence coverage, replicate precision, deuterium recovery, back-exchange control.

Overlay of isotopic envelopes for a peptide before and after deuterium labeling, with mass shift Δm ≈ 2.7 Da.

Representative isotopic envelopes showing a ~2.7 Da shift after deuterium labeling, indicating hydrogen–deuterium exchange.

Log-scale uptake curves of a peptide under two conditions (State A vs State B) with 95% CI shading, illustrating slower exchange in State B.

Deuterium uptake kinetics of a representative peptide, showing reduced uptake in State B compared to State A, consistent with structural protection.

Butterfly plot showing peptide-level ΔD values along the protein sequence, with threshold lines marking significant protection and exposure.

Sequence-resolved differential HDX plot; significant regions (|ΔD| ≥ 0.5 Da) highlight protected and exposed segments.

Protein 3D backbone colored by ΔD values, showing a protected binding interface and exposed flexible loops.

3D structural model with ΔD values mapped onto the backbone; blue regions indicate protection and red regions indicate exposure, highlighting a binding site.

Choosing Between HDX-MS and Other Protein Analysis Methods

Criterion	HDX-MS	Cryo-EM	X-ray	SPR/ITC/BLI
Best for	Local dynamics, binding footprints	Large complex architecture	Atomic detail of stable conformation	Affinity & kinetics
Resolution	Peptide → near-residue	Near-atomic	Atomic	Binding numbers only
Sample needs	Soluble, HDX-compatible	Homogeneous particles >150 kDa	Crystals required	Purified, sufficient material
Output	ΔD maps, kinetics	3D maps	Atomic coordinates	KD, kon/koff, ΔH

Case Study

HDX-MS Pinpoints the CD47–Nest1 Binding Interface

Title: The human CD47 checkpoint is targeted by an immunosuppressive Aedes aegypti salivary factor to enhance arboviral skin infectivity

Journal: Science immunology (2024) DOI: 10.1126/sciimmunol.adk9872

Objective

Identify the human target of mosquito salivary factor Nest1 and localize the binding epitope/conformational effects explaining enhanced ZIKV infectivity in skin.

Approach

REAP screening → CD47 hit; orthogonal biophysics (co-IP, SEC, SPR) plus HDX-MS for site-level mapping under solution conditions; functional assays in human immune cells and skin explants.

What HDX-MS revealed

Nest1 footprint: Residues 256–269 and 305–313 protected upon binding.
CD47 footprint: Residues 68–80 protected, overlapping the natural SIRPα site.
Quantitative: SPR Kd ≈ 38 nM for Nest1–CD47, ~25× tighter than human SIRPα (~1 μM).
Concordance: HDX-MS maps matched competition assays with high-affinity SIRPα variant CV-1.

Client value

Target validation: CD47 confirmed as the receptor engaged by Nest1.
Epitope-aware insight: HDX-MS enabled precise mapping to guide mutagenesis, blocking agents, or comparability studies.
Deliverables: ΔD tables, uptake kinetics, butterfly plots, 3D ΔD mapping, plus raw MS files and method details.

Nest1–CD47 binding characterized by SPR, domain mapping, and HDX-MS epitope protection

Biophysical characterization of the Nest1–CD47 interaction: SPR competition with CV-1, domain mapping of Nest1, and HDX-MS structural footprinting.

You May Want to Know

What can HDX-MS reveal about my protein?

It uncovers how proteins fold, move, and interact by mapping solvent accessibility and local dynamics, highlighting conformational changes and binding interfaces.

Is HDX-MS suitable for antibodies and ADCs?

Yes. It defines epitope/paratope regions, compares Fc/Fab stability, and assesses structural changes caused by conjugation in ADCs.

Can HDX-MS analyze membrane or difficult proteins?

With detergents, lipids, or amphipols, HDX-MS supports membrane proteins, receptors, and large complexes that are often inaccessible to other methods.

How does HDX-MS handle intrinsically disordered proteins?

It detects rapidly exchanging regions, distinguishing flexible loops and unstructured domains from stable folded areas in a single experiment.

Can HDX-MS study weak or transient interactions?

Yes. HDX-MS captures exchange kinetics, allowing detection of transient complexes and weakly bound states that traditional static methods often miss.

How reliable are HDX-MS results for decision-making?

Replicate variation is typically ≤10%, with strict temperature/pH control, statistical testing, and back-exchange benchmarks ensuring reproducibility.

How does HDX-MS compare with cryo-EM or X-ray?

Cryo-EM and X-ray deliver static atomic structures. HDX-MS provides dynamic solvent-accessibility data, making it a complementary tool for functional insights.

Can HDX-MS evaluate post-translational modifications (PTMs)?

Yes. By comparing modified vs. unmodified states, HDX-MS shows how phosphorylation, glycosylation, or conjugation affect local structure and stability.

Is there a size or complexity limit for HDX-MS?

No strict limit. HDX-MS is applied to antibodies, glycoproteins, multiprotein assemblies, membrane receptors, and even partially disordered proteins.

Do I need crystallization or isotope labeling for HDX-MS?

No crystallization or isotopic protein labeling is required; HDX uses D₂O exchange under solution conditions.