CreMap™ Epitope Mapping Service by Hydrogen/Deuterium Exchange

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.

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.

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.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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