Hydrogen/Deuterium Exchange based CreMap™ Epitope Mapping Service

Background Workflow Key Features Published Data FAQ Resources

Creative Biolabs has used hydrogen/deuterium (H/D) exchange on a research scale for many years and with the advancement of sample handling and data analysis software, now we are dedicated to offering our clients CreMap™ amide hydrogen/deuterium (H/D) exchange platform for epitope mapping.

Background

Hydrogen-deuterium exchange, also called H/D exchange is a powerful method for the identification of residues involved in the antigen-antibody interaction, which is a key step in understanding their functions, defining antibody specificity and predicting cross-reactivity. H/D exchange is a chemical reaction in which a covalently bonded hydrogen atom is replaced by a deuterium atom. It is usually used to exchangeable protons and deuterons.

For epitope mapping, H/D exchange is performed by exposing the peptide or protein of interest to the deuterated solvent for a period of time. The quenching in low pH buffer follows up where the exchange rates decrease dramatically. The proteins are then fragmented by the proteolysis of the protease. The epitopes are prevented from the proteolysis and protected by the antibody. These amino acids could be identified by mass spectrometry. H/D exchange is used as a powerful method and provides critical information needed to understand the mechanism of action of therapeutic antibodies. Mass spectrometry (MS) is widely used for defining epitopes due to effectiveness and its high resolution. The deuterium nucleus is heavier than the hydrogen nucleus as it contains a proton and a neutron. In a typical MS procedure, samples would be ionized and break into fragments. The fragments with or without deuterium would be separated according to the mass-charge ratio.

Fig.1 Hydrogen/Deuterium exchange of antigen for epitope mapping.

Workflow

The antibody is immobilized on media.
An antigen is mixed a deuterium buffer in solution, and the mixture was incubated for a series of predetermined time points.
The on-exchanged antigen is loaded onto an antibody column. The antigen-bound column is then washed with aqueous buffer.
The on/off exchange reaction is quenched by eluting out in a low pH buffer and digested by pepsin.
The percent deuterium incorporation into each peptide is measured by HPLC and MS.
The peptide with the deuterium is regarded as the epitope.

Key Features

Less sample amount required: H/D exchange method is based on mass spectrometry. Much fewer samples are needed compared with the conventional methods such as Nuclear Magnetic Resonance or X-ray crystallization. The concentration of samples could be as low as 0.1 µM.
Little size limitation: All the samples that are tested in H/D exchange go through the proteolysis process. The method could potentially be used for any peptide or protein.
Used for both linear and conformational epitopes: H/D exchange could be applied for identifying conformational epitopes as the atoms are “marked” by deuterium and mass spectrometry distinguishes and reveals the differences between hydrogen and deuterium.
High-resolution: Mass spectrometry is a technique that makes the chemicals ionized and engages the plot of the ion signal as a function of the mass-to-charge ratio with high precision. A particularly high mass resolution can necessarily resolve two peaks that have a certain small mass difference of at least 10ppm.

Creative Biolabs has massive experience on epitope mapping programs over the past few years and has been using latest mass spectrometry equipment and software to elucidate the binding sites. In addition, Creative Biolabs is working on optimizing CreMap™ H/D exchange workflow to provide top-quality service to our customers all around the world. If you are trying to figure out epitopes on the antigen in the study of an antibody-antigen complex, or if you are particularly interested in our H/D exchange platform, please contact us by sending E-mail. A formal feedback will be sent back as soon as possible. We are always more than ready to reach out.

Other optional CreMap™ B cell epitope mapping services:

Published Data

Identification of MAb1's Binding Epitope on TL1A by HDX-MS

Fig. 2 HDX revealed two major epitope regions of mAb1, peptide 1 and peptide 2. (Richard Y.-C. Huang, 2018)

The article elucidates the application of Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) in epitope mapping, specifically focusing on the interaction between an anti-TL1A monoclonal antibody 1 (mAb1) and the TL1A protein, a ligand for the death domain receptor DR3. This interaction is significant because TL1A, when bound to DR3, stimulates lymphocytes and triggers pro-inflammatory cytokines, making the blockade of this interaction a potential therapeutic strategy for autoimmune and inflammatory diseases. The study reveals that HDX-MS, coupled with computational modeling, identified a discontinuous epitope on TL1A that is targeted by mAb1. This epitope is within the predicted interaction interface of TL1A and DR3, suggesting that mAb1 blocks the TL1A/DR3 interaction through a unique mechanism, possibly by steric hindrance or direct interface blockade. This advanced mapping approach provides crucial molecular-level insights that are vital for the strategic development of targeted therapies against diseases involving TL1A/DR3 interactions.

References

Pandit, D., et al., Mapping of discontinuous conformational epitopes by amide hydrogen/deuterium exchange mass spectrometry and computational docking. J Mol Recognit, 2012. 25(3): p. 114-24.
Chalmers, M.J., et al., Differential hydrogen/deuterium exchange mass spectrometry analysis of protein-ligand interactions. Expert Rev Proteomics, 2011. 8(1): p. 43-59.
Huang, Richard Y-C., et al. "Hydrogen/deuterium exchange mass spectrometry and computational modeling reveal a discontinuous epitope of an antibody/TL1A Interaction." MAbs. Vol. 10. No. 1. Taylor & Francis, 2018.

FAQ

What is Hydrogen/Deuterium Exchange (HDX) used for in epitope mapping?
HDX is used in epitope mapping to identify the regions of an antigen that interact with antibodies. By measuring how the incorporation of deuterium into the antigen changes in the presence of an antibody, researchers can pinpoint the areas involved in binding, providing insights into the antibody-antigen interaction at a molecular level.
How does HDX work in the context of epitope mapping?
In HDX epitope mapping, the antigen is exposed to deuterium-enriched water. Regions of the protein involved in interactions, such as those with an antibody, will exhibit reduced deuterium uptake due to decreased solvent accessibility and/or changes in hydrogen bonding dynamics. Mass spectrometry is then used to measure the deuterium levels, indicating protected epitope regions.
What advantages does HDX offer over other epitope mapping techniques?
HDX provides detailed information about the conformational dynamics and solvent accessibility of proteins in solution, reflecting more natural conditions. It can differentiate between flexible and rigid parts of the antigen and is highly sensitive to changes in protein structure upon antibody binding, offering a comprehensive view of the epitope.
Can HDX identify both linear and conformational epitopes?
HDX is particularly effective for identifying conformational epitopes, which involve discontinuous amino acid sequences that come together in the three-dimensional structure of the protein. It can provide insights into how these complex structures are involved in antibody recognition, which is often not possible with techniques that only identify linear epitopes.
How does the presence of an antibody affect deuterium uptake in HDX?
When an antibody binds to its antigen, it typically protects certain regions of the antigen from solvent exposure. These regions, forming the epitope, will exhibit slower deuterium uptake compared to unbound states. Analyzing these differences allows researchers to identify which parts of the antigen are directly involved in binding.
How does HDX integrate with other analytical techniques for epitope mapping?
HDX is often combined with computational modeling, crystallography, or electron microscopy to provide a more comprehensive understanding of epitope structures. This integration allows for the validation of HDX results with structural data and can help in refining the models of how antibodies interact with antigens.
What role does HDX play in therapeutic antibody development?
HDX is crucial in the design and optimization of therapeutic antibodies. By identifying and characterizing epitopes, HDX helps in understanding the mechanism of action of antibodies and can guide modifications to improve binding affinity and specificity, ultimately enhancing therapeutic efficacy and reducing off-target effects.