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A free online tool for protein cleavage sites prediction with a chosen enzyme.
Calculate monoisotopic and average mass of a custom peptide or peptides predicted from a protein.
The cleavage specificities of selected enzymes.
A comprehensive and freely accessible resource of protein sequence and functional information.
Mass spectrum simulation tool for peptide/protein sequences. The number of charge can be custom defined.
An online tool predicts m/z values for SMS ions generated from selected fragmentation methods such as CID, ECD, or ETD. The ion type and maximum number of charge can be pre-defined.
PDB provides the most complete collection of information about the 3D structures of proteins, protein complexes and other biomolecules.
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What is Epitope mapping?
Epitope mapping is the process of identifying the binding site of an antibody on its target antigen protein. Epitope mapping is a crucial step during the development of new antibody drugs, vaccines, and diagnostics. It can help better understand the mechanism of how they function and develop more potent molecular candidates, and also provide required structural information for regulatory filling and patent protection.
Epitopes can be classified into two main groups: linear and conformational. Linear epitopes are composed of a sequence of continuous amino acids on just one segment of the antigen protein. In contrast, conformational epitopes are formed by amino acids from a number of discontinuous segments which are brought together upon three-dimensional protein folding. This character makes conformational epitopes more difficult to map because they are only formed in the native structure of the antigen, which cannot be reliably mimicked by using peptides. The vast majority of antibodies act through specific binding to the conformational epitopes on their antigen targets.
Epitope Mapping Methods
X-Ray Crystallography: It is a traditional method for structural biology. If appropriate crystals can be generated from the sample, it can provide single amino acid resolution data with high confidence, although flexible regions on the protein can be missed. However, this approach is technically challenging and is not always possible as crystal generation from antibody-antigen complexes tends to be difficult due to structural reasons (e.g. the diversity of PTMs and disordered regions in both the antibody and antigen). In addition, crystallography requires large amounts of purified protein, and can be time-consuming and expensive.
Peptide Scanning: This technique uses a library of synthetic peptides with overlapping sequences from a target protein, and analyzes their ability to bind the antibody of interest. The epitope mapping resolution here depends on the number of overlapping peptides used. This method is specifically suited to profile linear epitopes, but it is not possible for this technique to reliably map conformational epitopes because the peptides it relies on cannot adopt the same tertiary structure as that of the antigen protein.
Mutagenesis: Using this approach, specific amino acid residues of the antigen are mutated or changed followed by measurement of antibody binding to identify amino acids that interrupt the interaction. Because the mutation is often done by alanine replacement, this method is also called "alanine scanning". Mutagenesis offers the potential to give information at the amino acid level but suffers from incomplete epitope information and the uncertainty of not knowing whether an effect is direct or indirect due to an effect on the folding of a protein. Also, for conformational epitopes where a large interface is involved in the high affinity interaction, as in the case of most therapeutic antibodies, mutation of one residue in the epitope does not result in substantially decreased binding. These epitope residues will not be detected by this method.
Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS): This technique measures the solvent accessibility of amino acid residues in the protein using biological mass spectrometry, thus can determine the interaction site of the antigen-antibody complex in its native solution, and does not introduce any modifications (e.g. mutation) to either the antigen or the antibody. HDX-MS epitope mapping has also been demonstrated to be the effective method to rapidly supply complete information for epitope structure (http://www.pnas.org/content/110/9/3304). During analysis, both the antigen by itself and the antigen-antibody complex are incubated in deuterated solvent to allow the hydrogens to exchange with deuterium. Because the antibody provides extra protection for the residues in the binding site, the epitope can thus be determined by comparing the exchange behavior of the unbound antigen with that of the antigen-antibody complex. This technique works for both linear and conformational epitopes, but requires careful control of a series of experimental elements such as temperature, pH, exchange time, H/D back exchange and scrambling. The traditional “bottom-up” HDX-MS approach provides a peptide level resolution. NovoAb has developed an innovative FineMapping technology by using “top-down” HDX-MS and “middle-down” HDX-MS strategies, which enhanced the epitope mapping spatial resolution up to single amino acid. The unique subzero temperature UPLC technology used also provides unparalleled sensitivity by reducing the unwanted H/D back exchange from 30-50% to a minimal level (2%). This boosts the smaller HDX difference previously undetectable to above the detection limit of HDX-MS, and consequently leads to more accurate and complete mapping of the epitope.
