Molecular Characterization of a Conformational Epitope of Hen Egg White Lysozyme by Differential Chemical Modification of Immune Complexes and Mass Spectrometric Peptide Mapping

Abstract

A new approach for the characterization of conformationally dependent epitope structures in protein antigens is described using differential chemical modification of immune complexes in combination with mass spectrometric peptide mapping analysis. Well-established methods for epitope characterization are frequently not applicable to conformationally dependent epitopes, and direct methods of structure analysis such as X-ray crystallography of immune complexes have been successful only in a few cases. Our approach combines tertiary structure-selective chemical modification of immune complexes with the molecular characterization of reaction products by mass spectrometric peptide mapping. The comparison of the modification pattern of free and antibody-bound antigen provides the identification of residues protected from modification by the antibody. These residues hence are characterized as part of the epitope structure. The well-characterized hen egg white lysozyme and a corresponding monoclonal IgM-type antibody were investigated as a model system. Specific modification reactions for arginine, lysine, and tyrosine residues were performed, and the modification sites in free and antibody-bound antigen were determined by mass spectrometric peptide mapping. The R14 residue and residues K13 and K96 in the antibody-bound lysozyme were found to be protected from modification, comprising a surface of spatially adjacent residues by folding of the native protein. In contrast, other K and R residues as well as Y20 and Y23 showed no significant shielding from modification in the immune complex. These results provided an estimation of the molecular epitope surface area of native lysozyme.

Epitope Structure of the Carbohydrate Recognition Domain of Asialoglycoprotein Receptor to a Monoclonal Antibody Revealed by High-Resolution Proteolytic Excision Mass Spectrometry

“…Affinity mass spectrometry approaches using proteolytic epitope excision and epitope extraction have been previously described in detail and applied in epitope identifications [14][15][16][17][18][19]. In the general analytical method [14][15][16][17]19], the immobilised ligand-binder (antigen-antibody) complex is subjected to limited proteolytic digestion, followed by mass spectrometric analysis of the eluted affinity-bound epitope peptide fragment (s). In the proteolytic step, the epitope is protected from digestion owing to the shielding of the ligand-binder interaction structure; hence, epitope excision on an immobilised immune complex is essential for isolation, specific dissociation and identification of the bound epitope.…”

"Affinity-proteomics": direct protein identification from biological material using mass spectrometric epitope mapping

“…This characteristic feature of antigen-antibody complexes has been employed for the development of a general mass spectrometric approach for the identification of protein epitopes, as initially shown by combining limited proteolytic cleavage of intact immune complexes (epitope excision) with mass spectrometric peptide mapping [12]. Whereas initial applications of mass spectrometric epitope mapping have been performed on small sequence epitopes such as from pure polypeptides [15,17,18,19], several recent studies have shown that large, native proteins including conformational epitopes can also be successfully investigated [14,15,20,21,22,23,24,25]. Several bioanalytical applications have ascertained the mass spectrometric epitope mapping/epitope excision approach as a powerful tool; recent examples are the identification of a specific epitope from the Alzheimer's amyloid precursor protein, and the elucidation of an amyloid plaque-specific epitope which directly provides a lead structure for vaccine development against Alzheimer's disease [26,27].…”

Mass Spectrometric Characterization of a Discontinuous Epitope of the HIV Envelope Protein HIV-gp120 Recognized by the Human Monoclonal Antibody 1331A

“…Another approach for the characterization of discontinuous epitopes is differential chemical modification of amino acids in the immune complex and in the free Ag. Differences in the reactivity of the same amino acid in the Ab-Ag complex and the free Ag can provide information about the amino acids involved in affinity binding (37).…”