Epitope mapping has become one of the key elements in vaccine and drug development. Epitope mapping is the process of identifying the binding site of a protein to its corresponding antigen. FDA and EMEA guidelines recommend molecular profiling of interaction sites between a drug and its regulatory filing target. In addition, a better understanding of interaction sites can lead to the development of more effective drugs and patent-protected products. Epitope mapping, the process of identifying binding sites on an antigen to its corresponding antibody, is a key element in vaccine and drug development.
Epitopes vs. Paratopes. Epitopes are formed by amino acids on the target antigen, while paratopes are formed by amino acids on the binding antibody. Epitopes and paratopes interact to define the location and kinetics of binding.
Creative Proteomics' epitope mapping service has a success rate of more than 95%, and provides accurate, clear and directly applicable epitope definitions services for antibody projects of drug research and development companies and institutions.
Hydrogen-deuterium Exchange Mass Spectrometry (HDX-MS)
HDX-MS measures the accessibility of hydrogen molecules in the protein backbone. During the assay, both unbound antigen and bound antibody-antigen complexes are incubated in deuterium (D2O) water to exchange any amide hydrogens with deuterium from exposed amino acids of the protein backbone. The positions of these deuterium molecules on the protein sequence can then be carefully determined using high-resolution mass spectrometry. By comparing unbound antigen to bound antibody-antigen complexes, the residues of the epitope can be determined. The technique requires careful control of the temperature, pH and time of the reaction.
Utilizing high-resolution molecular structures through HDX analysis can also be used for the following projects:
Biosimilar Characterization
higher order structure
Conformation/Protein Dynamics
small molecule interaction
protein-protein interaction
Protein folding characterization
Cross-Linking Mass Spectrometry (XL-MS)
The ultra-large mass spectrometer first binds antibodies and antigens with mass-tagged chemical cross-linkers. Next, confirm the presence of the complex using high-quality MALDI detection. Since the Ab/Ag complex is very stable after application of cross-linking chemistry, multiple enzymes (five used in parallel) can be applied to the complex to provide many different overlapping peptides. Identification of these peptides was performed using high-resolution Orbitrap™ mass spectrometry and MS/MS techniques. Use a mass tag attached to the crosslinking reagent to determine the identity of the crosslinked peptide. After MS/MS fragmentation and data analysis using specific interaction software, epitopes and paratopes were determined in the same experiment. Due to the high sensitivity and accuracy of mass spectrometric detection, very small amounts of material are required.
Other applications of XL-MS:
protein-protein interaction
map aggregation
Paratope mapping analysis
Service Advantages
Rapid and stable detection of macromolecules and intact protein complexes by MALDI mass spectrometry
For all epitope mapping projects, individual proteins are first screened to confirm mass and detect significant multimerization. The intact complexes are then detected and stoichiometry is determined prior to epitope mapping.
Rapid screening saves our customers time and money.
Other Technologies
X-ray co-crystallization
X-ray co-crystallization is often considered the "gold standard" for epitope mapping. If successful, the technique could provide high-confidence resolution of single amino acids. However, this technique is not always feasible, as generating crystals from protein complexes is often difficult and in many cases impossible. This uncertainty often results in long project times. In addition, X-ray analysis requires large protein samples and is costly.
Peptide Scanning
In this technique, a series of overlapping peptides are generated from the antigen. These peptides are then analyzed to see if they disrupt the formation of the complex between the antibody and antigen. This is one of the most common techniques due to its relatively low cost and ability to quickly analyze large numbers of antibodies. This technique is mainly used for the analysis of linear epitopes.
| Item | Linear | Conformational | Complex Protein | High-resolution | Sample Consumption |
| XL-MS | ✔ | ✔ | 150ug | ||
| HDX-MS | ✔ | ✔ | 450 µg | ||
| X-ray co-crystallography | ✔ | ✔ | ✔ | tens of mg | |
| Peptide scanning | ✔ | ✔ | tens of µg |
Epitopes can be defined by two general categories, linear or conformational. A linear (or continuous) epitope consists of a linear stretch of amino acids in sequence, without three-dimensional structure. A conformational epitope is an epitope that requires tertiary folding to create a proper binding region.
Conformational epitopes most often occur in regions with breaks (discontinuities) in the amino acid binding sequence, requiring a folded structure to bring these regions close together. Conformational mapping techniques can be used to identify linear or conformational epitopes. However, linear techniques cannot map conformational epitopes.
Depending on the "resolution" of the technique used, the identified region can be a single amino acid, a small group of amino acids, or a peptide segment of a protein. In general, higher resolution techniques are more complex, time-consuming, and require more materials or time.
