Molecular characterization of a conformational epitope of hen egg white lysozyme by differential chemical modification of immune complexes and mass spectrometric peptide mapping.
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.
Fiedler
W
W
Faculty of Chemistry, University of Konstanz, Germany.
Borchers
C
C
Macht
M
M
Deininger
S O
SO
Przybylski
M
M
D000818:Animals; D000936:Antigen-Antibody Complex / Q000737:chemistry*; D002645:Chickens; D018360:Crystallography, X-Ray; D000939:Epitopes / Q000737:chemistry*; D005260:Female; D013058:Mass Spectrometry; D008958:Models, Molecular; D015394:Molecular Structure; D008970:Molecular Weight; D009113:Muramidase / Q000737:chemistry / Q000276:immunology*; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011487:Protein Conformation; D014357:Trypsin / Q000378:metabolism
D000936:Antigen-Antibody Complex; D000939:Epitopes; D010446:Peptide Fragments; D009113:Muramidase; D014357:Trypsin