Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Principles and Applications in Biomedicine

What is HDX-MS?

Hydrogen-deuterium exchange mass spectrometry is a mass spectrometry technique for studying the spatial conformation of proteins. The main principle of this technology is to put the protein in a heavy water solution, the hydrogen atom on the surface of the protein is exchanged with the deuterium atom in the heavy water, and the protein after the exchange is digested by enzymes to produce peptide fragments. Mass spectrometry identifies the quality of peptide fragments: Peptides located on the protein surface are more likely to undergo hydrogen and tritium atomic exchange than peptides located in the protein interior, thus predicting the protein epitope conformation. In addition, hydrogen-deuterium exchange mass spectrometry can also be used to study the dynamic changes of protein structure, and protein interaction sites, and identify protein surface active sites.

Hydrogen-deuterium exchange mass spectrometry can quickly study the amino acid sequences on the surface of natural proteins in solution, so it has a wide range of applications in the study of protein dynamic epitope active sites. Due to the principle of hydrogen-deuterium exchange mass spectrometry, the backcross of hydrogen-deuterium atoms has a great impact on the accuracy of experimental results, so it is very important to avoid backcross of hydrogen-deuterium atoms. At present, the control of hydrogen-deuterium atomic backcross is mainly through shortening the liquid-mass analysis time and keeping the temperature and pH within the range of the lowest backcross reaction coefficient.

Principles of HDX-MS

The core principle of HDX-MS is based on the dynamic exchange reaction between amide hydrogen (N-H) and deuterated solvent (D₂O) in the main chain of the protein. In solution, the conformational dynamics of proteins (such as folding, binding, or dissociation) directly affect the solvent accessibility and hydrogen bond stability of their local structures: exposed flexible regions are highly labeled due to fast deuteration rates, while protected stable regions (such as alpha helix, beta folding) are significantly reduced due to H-bond network shielding. By controlling the reaction time and using high-resolution mass spectrometry to accurately detect small changes in the molecular weight of the peptide (1 Da per addition of a deuterium atom), HDX-MS is able to dynamically transform the conformation into a quantifiable deuterium map, revealing protein interaction sites, dynamic structural changes, and functional regulatory mechanisms. This technique directly analyzes natural proteins under physiological conditions without the need to crystallize or fix samples, becoming a key tool for resolving the "dynamic behavior" of biological macromolecules.

Features of HDX-MS

• High Sensitivity: Detects subtle conformational changes and protein-ligand interactions.
• Minimal Sample Requirement: Requires low protein concentrations, making it suitable for limited samples.
• Broad Applicability: Works with various biomolecules, including proteins, protein complexes, and nucleic acids.
• Native Condition Analysis: Provides insights into protein behavior under physiological conditions.
• Structural Resolution: Identifies flexible and solvent-accessible regions at peptide or residue-level resolution.
• Real-Time Kinetics: Captures dynamic changes in protein conformation over different time scales.

What is HDX Used For?

• Protein Conformation Analysis: Identifies structural changes, folding states, and disorder regions.
• Protein-Ligand Interactions: Maps binding sites and conformational shifts upon ligand binding.
• HDX-MS Epitope Mapping: Determines antibody-antigen interactions for vaccine and therapeutic development.
• Protein Complex Assembly: Studies subunit interactions and allosteric effects.
• Comparative Structural Studies: Analyzes wild-type vs. mutant proteins or different formulation conditions.

Chemical Mechanism of Hydrogen-Deuterium Exchange

Chemical Basis of Amide Hydrogen Exchange

The Chemical Nature of Exchange Reactions

The amide hydrogen (-NH-, located in the peptide bond) on the protein backbone undergoes a reversible hydrogen-deuterium isotope exchange reaction in solution with solvents such as D₂O. The reaction is essentially an acid-base catalytic process:

Protonation and Deprotonation

The exchange of amide hydrogens undergoes a transition state of protonation (N-H → N-H⁺) and deprotonation (N-H⁺ → N⁺ + H⁺). In D₂O, deuterium ions (D⁺) replace H⁺, eventually forming the N-D bond.

Figure 1. Overview of the basic principles of amide exchange in proteins.: (A) The protein samples a closed (cl) and open (op) forms, of which only the open form is accessible for deuterium exchange. The relative rates of opening (k_op), closing (k_cl), and the chemical exchange rate of the accessible amide (k_ch) govern whether the experimentally observed rates will fall in the realm of (B) EX1 kinetics or (C) EX2 kinetics. (D) For EX2 kinetics, the free energy of the local stability can be calculated using the ratio of the k_ch and the observed rate k_obs, a ratio referred to as the protection factor (PF) associated with the local structure. (James EI,2022)

Indicative Role of Dynamic Structure

Exchange Rate Mapping Structure Features

• Fast switching area (milliseconds):
  Corresponding disordered regions, flexible rings, or surface exposed areas indicate a dynamic or unfolded state (such as a flexible gated structure near an enzyme active site).
• Slow switching area (minute to hour level):
  Corresponding to stable secondary structures (such as α-helix cores) or intermolecular interaction interfaces (such as antibody-antigen binding epitopes), reflecting structural rigidity or binding protection effects.

Dynamic Tracking of Conformational Change

• Ligand binding analysis:
  When proteins bind to small drug molecules or biological macromolecules (such as RNA, and DNA), the exchange rate of amide hydrogen near the binding site is significantly reduced due to spatial shielding or hydrogen bond strengthening. By comparing the deuteration difference before and after binding, the binding site can be accurately located.
• Case in point:
  After the kinase binds to an ATP analog, the deuteration rate of the peptide around its active pocket decreases by 30%, suggesting conformational closure caused by binding.
• Folding and unfolding monitoring:
  During the protein folding process, the deuteration rate of the core hydrophobic region decreased gradually with time, indicating that the structure was gradually stable. Refolding (such as under denaturation conditions) results in a rapid increase in deuteration rate.
• Allosteric effect analysis:
  Remote conformational changes induced by allosteric regulators can be captured by deuteration rate changes in distal peptide segments (such as dynamic transmembrane transport after GPCR ligand binding).

Time-resolved dynamic processes

By controlling the deuterium labeling time (e.g., 10 seconds, 1 minute, 10 minutes), the transient intermediate state can be distinguished from the stable state:

• Short labeling time: Captures fast dynamic events (such as instantaneous binding of a protein to a cofactor).
• Long labeling time: reveals overall conformational stability (e.g. antibody thermal stability assessment).

Experimental Workflow of HDX-MS

All HDX-MS experiments require deuterium labeling prior to MS analysis. The protein is incubated in a deuterium buffer so that the amide hydrogen present on the protein backbone can be exchanged with the deuterium buffer. The most common labeling method is continuous labeling, in which proteins in a stable state are incubated continuously in a deuterium buffer for different time periods and the exchange of hydrogen with deuterium is measured as a function of time. Time periods can range from seconds to hours or days. After labeling, the experimental temperature was reduced to 0°C, and the pH of the reaction was reduced to 2.5 to quench the sample. HDX-MS experiments can be conducted either bottom-up or complete/top-down.

HDX-MS is a highly standardized, multi-step process designed to precisely capture dynamic conformational changes in proteins.

Deuteration

Deuterium labeling is the first step of the HDX-MS experiment, the core of which is to place the protein sample in a buffer containing deuterated solvent (D₂O) so that the amide hydrogen (N-H) on the protein main chain is dynamically exchanged with deuterium (D). The key to this process is to control labeling time (from seconds to hours) and reaction conditions (e.g., pH, temperature) to capture dynamic conformational changes on different time scales. For example, a short time marker (10 seconds) captures an instantaneous intermediate state, while a long-time marker (1 hour) reflects a stable conformation. During the labeling process, the flexible regions exposed to the solvent are highly labeled due to the fast deuteration rate, while the protected stable regions (such as α-helix and β-fold) are significantly reduced due to hydrogen bond shielding. By optimizing the labeling conditions, the researchers were able to accurately capture the dynamic behavior of the protein, laying the foundation for subsequent analysis.

Quenching

Quenching is a key step in HDX-MS experiments designed to terminate the hydrogen-deuterium exchange reaction by rapidly reducing pH and temperature, locking in the current deuterium state. Quenching is usually performed in an ice water bath (0-4 °C) with the addition of a pre-cooled acidic buffer (such as formic acid or trifluoroacetic acid at pH 2.5) to reduce the pH of the solution to 2.0-2.5, thus inhibiting further exchange of the amide hydrogen. In addition, some protocols include denaturants (such as urea or guanidine hydrochloride) to completely destroy the secondary structure of the protein and prevent local residual exchange. The quenching process is completed within a few seconds to ensure the accuracy and repeatability of the data. The success of this step directly determines the resolution and reliability of subsequent mass spectrometry.

Digestion & Separation

Enzymatic hydrolysis and separation are the key steps in converting proteins into short peptide segments suitable for mass spectrometry analysis. First, the quenched sample is enzymolized by Pepsin to produce a 5-20 amino acid peptide. Pepsin is preferred because of its high activity at low pH and its wide range of cutting sites. Enzymolysis can be performed online (via immobilized enzyme columns) or offline (static digestion) mode, with the former being more suitable for high-throughput analysis. Subsequently, the enzymolysis products were separated by reversed-phase liquid chromatography (LC) using C18 columns and acetonitrile/formic acid gradient elution to reduce peptide co-elution and improve the specificity of mass spectrometry detection. Optimization of this step, such as gradient slope adjustment, is critical to improving data quality.

MS Analysis & Data Processing

Mass spectrometry and data processing is the last step of HDX-MS experiment, and also the core of data generation. High-resolution mass spectrometry (such as Orbitrap or Q-TOF) is used to detect the mass shift of the peptide segment, calculating the amount of deuterium atom incorporation from the isotopic peak distribution (1 Da per deuterium generation). The first step of data analysis is to use software (such as Byonic, and Peaks) to match the original mass spectrometry data to the theoretical enzymatic hydrolysis peptide library and identify the peptide sequence. Subsequently, the relative deuterium quantities (comparing the mass shift of the experimental group with the unlabeled/fully deuterated control) were calculated and the time-resolved deuterium curves were fitted to reveal the dynamic conformational changes. Finally, the deuterium differences are mapped to the three-dimensional structure of the protein (such as a PDB file), producing a visual heat map that visually shows the dynamic regions. The precision and depth of this step directly determine the scientific value of the HDX-MS experiment.

Learn more about HDX-MS Workflow from Sample Preparation to Data Interpretation.

Characteristics and Advantages of HDX-MS

Compared with Traditional Technology

As a new dynamic structure analysis technology, HDX-MS has shown unique advantages over traditional methods such as X-ray crystallography and nuclear magnetic resonance spectroscopy (NMR) in many aspects.

Resolution and Information Dimension

• HDX-MS:
  Spatial resolution: It can be located to the level of a single peptide (5-20 amino acids), and amino acid residue resolution can be reached after optimization of some technologies.
  Time resolution: The millisecond time scale captures dynamic processes (such as protein folding, and ligand binding transient intermediate states).
  Information dimension: provides a panoramic map of dynamic conformational changes, and combines mass spectrometry data with structural models to achieve multidimensional analysis.
• X-ray crystallography:
  Spatial resolution: Atomic resolution (<2 A), but limited to static crystal structure.
  Temporal resolution: cannot capture dynamic processes and only reflects a single conformational state.
  Information dimension: static structure information, lack of dynamic behavior description.
• NMR:
  Spatial resolution: Atomic resolution, but limited to small molecular weight proteins (<50 kDa).
  Time resolution: milliseconds to seconds, can partially capture the dynamic process, but the sensitivity is low.
  Information dimension: provides local dynamic information, but has limited ability to resolve macromolecular complexes.

Scope of Application and Sample Requirements

• HDX-MS:
  Sample type: Compatible solution protein, membrane protein, post-translational modified protein, and macromolecular complex (>150 kDa).
  Sample size: Only microgram sample (μg), suitable for precious or low abundance proteins.
  Sample state: No need to crystallize fix, maintain the natural conformation.
• X-ray crystallography:
  Sample type: High-quality crystals are required, and it is difficult to crystallize membrane proteins, flexible proteins, and macromolecular complexes.
  Sample size: A Milligram sample (mg grade) is required, the crystallization process is time-consuming and the success rate is low.
  Sample state: static crystal structure, may deviate from physiological state.
• NMR:
  Sample type: Limited to small molecular weight proteins (<50 kDa), not applicable to membrane proteins and macromolecular complexes.
  Sample size: milligram sample (mg grade) and isotope labeling (e.g. ¹³C, ¹⁵N) is required.
  Sample state: Solution state, but high concentration requirements may lead to aggregation.

Biomedical Application Scenarios

HDX-MS is widely used in biomedical research because of its high sensitivity and dynamic resolution ability.

Drug Development

Differential HDX-MS analysis of proteins under different conditions, such as apoproteins versus holoproteins, has become an important tool for exploring the effects of chemical modifications, mutations, and binding events on protein stability and conformational dynamics. Recently, HDX-MS-guided drug design was used to develop SR1664, a representative molecule of a novel functionally selective PPARγ modulator (FSPPARM) that acts as a classical antagonist while regulating the phosphorylation of obesity-induced receptor and target gene subsets.

Antibody Engineering

HDX-MS is a powerful technology that is increasingly being used to study protein-protein interactions. It can be combined with computational protein modeling schemes to generate high-resolution near-natural models. Tran MH et al. discuss how HDX-MS can be applied to map Ab-Ag interactions and argue that HDX-MS is a very efficient and fast method for studying Ab-Ag complexes as well as proteins that are not suitable for other structural methods due to their inherent properties.

Disease mechanism

HDX-MS can provide important support for the study of disease mechanisms, mainly due to its unique advantages in the analysis of protein dynamic conformation. Rider MH et al. used HDX-MS to investigate the molecular mechanisms of AMPK activation and thermal proteomic analysis (TPP) to assess the pharmacological off-target effects of AMPK activators/inhibitors.