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CreMap™ Epitope Mapping by Limited Proteolysis Coupled with Mass Spectrometry
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.
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
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The antigen is bound to the immobilized antibody, followed by digestion with various proteases.
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Proteases cleave the antigen at different sites, are used for the digestion.
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Unbound peptides are washed off the column and the affinity-bound peptides are analyzed using mass spectrometry.
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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
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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.
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Low sample consumption: Our instruments are sensitive and a few samples are required for mass spectrometry.
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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.
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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.
References
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Zhao, Y. and B.T. Chalt, Protein epitope mapping by mass spectrometry. Anal Chem, 1994. 66(21): p. 3723-6.
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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.
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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.
All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.