The ability to accurately characterize an epitope on an antigen is essential to understand the pathogenesis of an infectious material, and for the design and development of drugs and vaccines. Emergence of a new contagious microbial or viral variant necessitates the need for robust identification and characterization of the antigenic determinant. Recent advances have made mass spectrometry (MS) a robust and sensitive analytical tool with high mass accuracy. The use of MS to characterize peptides and proteins has gained popularity in the research arena involving protein-protein interactions. Combining the modern mass spectrometric principles of protein-protein interaction studies with the classical use of limited proteolysis, a linear epitope on a peptide or a protein antigen can be accurately mapped in a short time, compared with other traditional techniques available for epitope mapping. Additionally, complete MS analyses can be achieved with very little sample consumption. Here we discuss the overall approach to characterize the detailed interaction between a linear antigen (either a peptide or a protein antigen) and its corresponding monoclonal antibody by using MS. The steps involved in epitope excision, epitope extraction, and indirect immunosorption are outlined thoroughly. Conditions required for MS analysis using either matrix assisted laser desorption ionization (MALDI) or electrospray ionization (ESI) sources are summarized, with special emphasis on the experimental protocols.
"As golden standards of epitope characterization these methodologies allow precise identification of the amino acid side chains involved in binding, but they are not suited for large-scale epitope identification and their results cannot be interpreted readily in terms of possible cross-reactions. Other epitope mapping approaches include proteolytic fragmentation , analysis of protein arrays and peptide arrays , or analysis of recombinant antigen (including antigens arrayed by in situ cell-free translation , mutagenized  and/or expressed using selectable systems such as phage display ). Despite this plethora of epitope-mapping methods, detailed epitope information lacks for the vast majority of antibodies used in life science research. "
[Show abstract][Hide abstract] ABSTRACT: We have recently developed a high-density photolithographic, peptide array technology with a theoretical upper limit of 2 million different peptides per array of 2 cm(2). Here, we have used this to perform complete and exhaustive analyses of linear B cell epitopes of a medium sized protein target using human serum albumin (HSA) as an example. All possible overlapping 15-mers from HSA were synthesized and probed with a commercially available polyclonal rabbit anti-HSA antibody preparation. To allow for identification of even the weakest epitopes and at the same time perform a detailed characterization of key residues involved in antibody binding, the array also included complete single substitution scans (i.e. including each of the 20 common amino acids) at each position of each 15-mer peptide. As specificity controls, all possible 15-mer peptides from bovine serum albumin (BSA) and from rabbit serum albumin (RSA) were included as well. The resulting layout contained more than 200.000 peptide fields and could be synthesized in a single array on a microscope slide. More than 20 linear epitope candidates were identified and characterized at high resolution i.e. identifying which amino acids in which positions were needed, or not needed, for antibody interaction. As expected, moderate cross-reaction with some peptides in BSA was identified whereas no cross-reaction was observed with peptides from RSA. We conclude that high-density peptide microarrays are a very powerful methodology to identify and characterize linear antibody epitopes, and should advance detailed description of individual specificities at the single antibody level as well as serologic analysis at the proteome-wide level.
PLoS ONE 07/2013; 8(7):e68902. DOI:10.1371/journal.pone.0068902 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The identification of antigenic epitopes is important for the optimization of monoclonal antibodies (mAbs) intended as therapeutic agents. MS has proven to be a powerful tool for the study of noncovalent molecular interactions such as those involved in antibody-antigen (Ab-Ag) binding. In this work, we described a novel methodology for mapping a linear epitope based on direct mass spectrometric measurement of Ab-Ag complexes. To demonstrate the utility of our methodology, we employed two approaches, epitope excision and epitope extraction, to study a model system consisting of a Fab antibody fragment with specificity toward the peptide abeta(1-40). In epitope excision, the Fab and abeta(1-40) complex was treated with proteolytic enzymes and the digested complexes were directly monitored by MS under native conditions. Mass differences between the Fab-abeta complex and the Fab control revealed the size of epitope peptides that were bound to the Fab. Using the epitope extraction approach, abeta(1-40) was first digested by Lys-C, and the fragment containing the epitope was selected by Fab binding. Data analysis allowed mapping of the epitope to abeta(16-27) which is in good agreement with previously unpublished data. The utility of the methodology was demonstrated by elucidating the binding epitopes for two full-length anti-abeta(1-40) mAbs.
[Show abstract][Hide abstract] ABSTRACT: The authors describe a technique for mapping the epitopes of protein antigens recognized by mono- or polyclonal antibodies. This method is based on a recombinant polypeptide library, expressed in a bacterial expression system, arrayed at high density, and tested on a membrane with automated procedures. The authors analyzed the epitope of a commercially available monoclonal antibody to vitamin D receptor (VDR). About 2300 overlapping VDR peptides were screened on a test array, and a contiguous stretch of 37 amino acids was identified as the epitope. Its authenticity was confirmed by Western blotting and an immunofluorescence competition assay on human skin tissue samples. The authors define the proposed method as a cell-based protein or peptide array that is adaptable to many applications, including epitope mapping of antibodies and autoantibodies, autoantigen detection from patient sera, whole-proteome approaches such as protein-peptide interactions, or selection of monoclonal antibodies from polyclonal sera. The advantages of this method are (a) its ease of protein array production based on well-established bacterial protein/peptide expression procedures; (b) the large number of printable colonies (as many as approximately 25,000) that can be arrayed per membrane; (c) there is no need for protein purification of recombinantly expressed proteins; (d) DNA, rather than protein, is the starting material to generate the arrays; and (e) its high-throughput and automatable format.
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