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Epitope Mapping by Crosslinking Mass Spectrometry (XL-MS)
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CovalX offers a unique analytical service for epitope mapping based on our patented technology. Our service allows conformation or linear epitope determination of monoclonal antibodies targeting any type of protein antigen, with no limit on molecular weight. cross-linking provides detailed structural information on antibody-antigen interactions, enabling paratope identification and direct insights into interaction stoichiometry, aggregation, and antibody integrity.
Epitope Mapping by XL-MS Step by Step
Leveraging High Mass MALDI MS technology and crosslinking expertise, CovalX offers a meticulous epitope mapping process:
- Sample stabilization through covalent crosslinking.
- Precise epitope characterization via MS and covalent tagging, achieving 3-4 amino acid resolution.
- Accurate identification of interacting peptides without peptide synthesis. This method is cost-efficient and time-effective, with optimized digestion conditions.
Step 1: Initial Sample Screening
Before the high-resolution analysis of the epitope and paratope can begin, CovalX performs High-Mass MALDI analysis on the antibody, antigen and the intact antibody/antigen complex.
The goals of these analyses are to verify:
- The integrity of both the antibody and the antigen
- The possible aggregation of the antibody
- The possible multimerization of the antigen
- The stoichiometry of the antibody/antigen interaction (monovalency, bivalency, etc)
- Verification that the specific intact cross-link antibody/antigen complex is present in the sample before proteolytic cleavage
Step 2: Crosslinking Mass Spec Mapping (XL-MS)
After initial sample screening is completed, an individual length of cross-linker is selected which is shown to effectively stabilize the complex using our high mass MALDI detection. A 50:50 mixture of deuterated to undeuterated cross-linker is created to provide a unique mass tag for detecting the linkers location within the proteins sequence. CovalX uses unique software to later detect these deuterated cross-linkers directly, allowing the highest degree of confidence in the data.
Step 3: Multiple Enzymes Provide Highest Coverage
After stabilization, four different enzymes are utilized in parallel for four separate and comprehensive digestions. Trypsin, Chymotrypsin, Elastase, and Thermolyse are each utilized individually to ensure the most thorough detection possible. The peptide coverage from the sum of these digestions provides the highest sequence coverage available with the most overlapping possibilities.
Finally, the peptide data is matched to the sequences using dedicated software to ensure proper identification of both the paratope and epitope from one data set.
scientist on the phone Timeline, Sample Requirements & Resolution
Our XL-MS epitope mapping services offer a timeline of 4-5 weeks for one epitope (shorter with RUSH) and 5-6 weeks for projects with up to 5 epitopes targeting the same antigen. Sample requirements include 200 μg of antigen and 200 μg of mAb/epitope, with a resolution down to 3-4 amino acids or less. Suitable for conformational, discontinuous, and linear epitopes, applicable to any therapeutic proteins or antigen with no size or complexity limits.
Why is CovalX’s Epitope Mapping Services the best Option?
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.
| Readout | Characterization of conformational or linear epitopes |
|---|---|
| Determination of both epitope and paratope | |
| Affordable | Competitive cost compared to other mapping techniques |
| Low Sample Consumption | Only 200μg of each antigen/antibody required |
| Full Compatibility | No need for immobilization, recombinant protein work, peptide synthesis or micro-array. The interaction is analyzed directly by Mass Spectrometry in solution. Analysis of full mAbs, fAbs, tagged proteins or modified proteins. |
| Fast Turnaround | Four to five weeks delivery time (less with RUSH) |
| Quality Work | Expert team with more than 15+ years of epitope mapping experience using mass spectrometry. |
Related Services
Download supporting materials
CovalX Crosslinking Mass Spec Mapping (XL-MS) Brochure
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.