Limited Proteolysis & Mass Spectrometry based CreMap™ Epitope Mapping Service

Background Workflow Key Features FAQ Resources

Creative Biolabs provides epitope mapping services by using limited proteolysis coupled with the most advanced mass spectrometry equipment. The enzymes used in our platform provide highly optimized digestion conditions with the highest sequence coverage possible. Creative Biolabs is confident to find the epitope region with high accuracy and resolution.

Background

Accurately characterizing epitopes on an antigen is a critical step for understanding the pathogenesis of an infectious material, and for the development of drugs and vaccines against toxins or enzymes. Proteolytic fragmentation has been used for mapping protein epitopes of antigens and proteolytic cleavage of antigen-antibody complexes provides a simple, cost-effective and straightforward approach to characterize a linear epitope. Proteins are cleaved by enzymes with different restriction sites to generate overlapping peptides. These peptides are then separated and identified by Mass Spectrometry (MS). The application of MS to characterize peptides and proteins has become a versatile and powerful method to precisely determine a soluble antigen’s partial structure (epitope) which interacts with the antibody. By comparing free antigen and antigen-antibody complex, a linear epitope on the antigen can be accurately mapped in a short time. Over the years this method has been established as a reliable source of data for both research and therapeutic purposes.

Fig.1 Scheme of the epitope mapping method by limited proteolysis.

Workflow

The antigen is bound to the immobilized antibody, followed by digestion with various proteases.
Proteases cleave the antigen at different sites, are used for the digestion.
Unbound peptides are washed off the column and the affinity-bound peptides are analyzed using mass spectrometry.
Comparison of elution profiles with and without the antibody showed a shift only for epitope-bearing peptides, enabling direct identification of the epitope(s).

Key Features

More applicable, especially for linear epitopes: The application of mass spectrometry has triggered the development of identification of epitopes. For limited proteolysis, it is advantageous when the goal is to determine the smallest number of amino acid residues.
Low sample consumption: Our instruments are sensitive and a few samples are required for mass spectrometry.
Few handing steps: Proteolysis is one of the simplest methods available for identifying epitopes. Mass spectrometry has easier handling preparation methods compared with Nuclear Magnetic Resonance or X-ray crystallization.
High specificity and precision and Short duration of analysis: 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. Due to its relatively simple operation, mass spectrometry is more time-efficient than the conventional methods such as Nuclear Magnetic Resonance or X-ray crystallization.

Creative Biolabs has accumulated extensive experience to offer epitope mapping services. Our experienced scientists will assist you in designing the research program that best suits your purpose and advances your research. If you are now trying to figure out epitopes on an antigen, or if you are interested in our platform, please contact us. 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:

References

Zhao, Y. and B.T. Chalt, Protein epitope mapping by mass spectrometry. Anal Chem, 1994. 66(21): p. 3723-6.
Pimenova, T., et al., Epitope mapping of bovine prion protein using chemical cross-linking and mass spectrometry. J Mass Spectrom, 2008. 43(2): p. 185-95.
Suckau, D., et al., Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping. Proc Natl Acad Sci U S A, 1990. 87(24): p. 9848-52.

FAQ

What is epitope mapping by limited proteolysis and mass spectrometry?
Epitope mapping by limited proteolysis and mass spectrometry involves digesting proteins with specific proteases under controlled conditions, then identifying the resulting fragments using mass spectrometry. This method helps pinpoint protein regions (epitopes) that are involved in interactions with antibodies or other molecules, based on which fragments are protected or exposed.
How does limited proteolysis contribute to epitope mapping?
Limited proteolysis selectively breaks down protein regions that are accessible and flexible, leaving structured or bound regions (like those involved in binding to antibodies) intact. Analyzing which parts of the protein are digested and which are not provides clues about where epitopes are located on the protein surface.
What role does mass spectrometry play in this process?
Mass spectrometry accurately identifies and quantifies the peptides resulting from proteolysis. It measures the mass-to-charge ratio of ions to identify the molecular structure of peptides, helping researchers determine which parts of the original protein were protected due to binding interactions.
What are the advantages of using this method for epitope mapping?
This technique is highly sensitive and can provide detailed information about the primary structure of protein epitopes. It is effective even with complex mixtures of proteins and can distinguish between closely related peptides, offering high-resolution insights into protein-protein interactions.
Can this method differentiate between different types of epitopes?
Limited proteolysis coupled with mass spectrometry can differentiate between linear and discontinuous epitopes. By examining the pattern of peptide fragments, researchers can infer whether epitopes are composed of contiguous amino acid sequences or if they are made up of segments brought together by protein folding.
What are the limitations of epitope mapping by this method?
This method may not effectively identify epitopes that involve large or complex conformational structures unless those structures influence the protein's susceptibility to protease digestion. Additionally, the interpretation of mass spectrometry data requires sophisticated software and expertise in protein chemistry.
How does this method compare to other epitope mapping techniques?
Compared to methods like X-ray crystallography or NMR spectroscopy, limited proteolysis and mass spectrometry is less labor-intensive and does not require crystallization or isotopic labeling. However, it provides less direct information about the spatial arrangement of atoms within the epitope compared to these more direct structural methods.
What improvements have been made in this method recently?
Recent improvements include the use of more specific and varied proteases to achieve finer control over digestion, as well as advances in mass spectrometry technology that improve sensitivity and accuracy. Enhanced computational tools for analyzing mass spectrometry data have also been developed, helping to better predict and characterize epitopes.
What applications does this method have outside of epitope mapping?
Beyond epitope mapping, limited proteolysis and mass spectrometry are used in studying protein folding, protein-protein interactions, and protein stability. It's also utilized in quality control for therapeutic proteins and vaccines to ensure that correct structural conformations are maintained, which is crucial for their efficacy and safety.