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Epitope Mapping by Hydrogen Deuterium eXchange Mass Spectrometry (HDX-MS)
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HDX-MS is a powerful method for mapping both linear and conformational epitopes. Widely acknowledged in proteomics for its ability to capture the dynamic nature of conformational epitopes and their interactions with antibodies or ligands, HDX-MS is also useful for identifying epitopes requiring tertiary folding or structural rearrangements. CovalX’s HDX-MS epitope mapping services boast high resolution and success rates, supported by advanced equipment and experienced technicians proficient in optimizing HDX-MS workflows for precise epitope characterization.
The Principle of HDX-MS
The principle of HDX-MS involves the labeling of proteins or peptides with deuterium, followed by their exposure to solvent. The extent of deuterium incorporation is then measured using mass spectrometry, revealing information about protein structure and dynamics. Originally for academia and pharmaceuticals, automation and data advances broaden its accessibility, slashing time and sample needs. Now, HDX-MS is viable for mainstream use, revealing protein dynamics with efficiency and precision.
Key Advantages of HDX-MS:
- Provides insights into conformational and linear epitopes
- Requires low sample consumption
- Offers high resolution, down to single amino acid level
- Applicable for a wide range of proteins and antigens
- Eliminates the need for crystallography or recombinant protein work
- Cost-effective compared to alternative mapping techniques
Step 1: Initial Sample Screening
Before the high-resolution analysis of the epitope begins, CovalX first performs High-Mass MALDI analysis on the intact antibody and antigen. This initial screening utilizes CovalX exclusive High-Mass MALDI detections systems to ensure that the HDX experiment is performed on characterized and controlled proteins.
This analysis is unique to CovalX services. The goal of this analysis is to verify:
- The integrity of both the antibody and the antigen
- The possible aggregation of the antibody
- The possible multimerization of the antigen
Step 2: Hydrogen Deuterium eXchange (HDX-MS)
When diluted in heavy water (D2O), backbone hydrogens of amino acids exchange with deuterium at varying kinetics rates depending upon their hydrogen bonding and solvent accessibility. When the antigen is complexed with the antibody, this deuterium uptake rate is altered in the epitope regions. These differences can be measured accurately at various time points.
After initial screening, the unbound antigen and the antibody-antigen complex are diluted in a D2O solution
At various time points, the Deuterium/Hydrogen exchange is quenched at 0°C and pH 2.5
The protein samples are then digested into peptides using appropriate proteolytic digestion
The resulting peptides are then directly injected for microflow LC-MS/MS detection
From the peptide mass fingerprints (PMF), deuterium exchange rate heat maps can be compared between the antigen alone or the antigen with antibody binding
Step 3: Data Analysis and Report
Finally, data is analyzed using the latest software packages from multiple providers with full report generation using easy to understand HDX heat map descriptions and personal result presentation. Graphical characterization upon the PDB structure included as available.
HDX analysis Timeline, Sample Requirements & Resolution
CovalX offers the lowest cost for HDX analysis. Our HDX-MS epitope mapping services require 400 ug of antigen and 400 ug of mAb/epitope, with resolution down to 4-8 amino acids. Delivery time is 5-6 weeks, less with RUSH service. It’s applicable for most therapeutic proteins or antigens and eliminates the need for crystallography, recombinant protein work, peptide synthesis, or micro-array.
Why Choose CovalX’s Epitope Mapping Services by HDX-MS?
CovalX offers two techniques for conformational epitope mapping by mass spectrometry: Hydrogen Deuterium eXchange (HDX-MS) and Crosslinking Mass Spectrometry (XL-MS). Each of these technologies provides unique advantages.
Notable Benefits with CovalX HDX-MS Service
| Technique | Sample handling is conducted using the latest automation robotics. Mass spectrometry and data analysis with the latest instrumentation and software |
|---|---|
| Expertise | Experienced epitope mapping team servicing the industry for more than 15 years |
| Quality Work | Unique mass spectrometric characterization of the intact proteins (aggregation, multimers, stoichiometry of the protein complexes) before starting the HDX-MS experiments. Our pre-HDX sample understanding provides the most reliable rate of success |
| Affordable | Most competitive technique cost/resolution/timeline |
| Low Sample Consumption | Only 300 μg of each antigen/antibody required |
| Fast Turnaround | 4 weeks (or less with RUSH) delivery time |
Other Services that CovalX HDX-MS provide
Download supporting materials
Hydrogen Deuterium eXchange Mass Spectrometry Services (HDX-MS)
For example patents, publications, or comparison data please see our library or contact us directly.
An overview of epitope mapping technologies can be seen here.