Mass spectrometric mapping of protein epitope structures of myocardial infarct markers myoglobin and troponin T.
Monoclonal antibodies are widely used analytical tools in biochemical research. The knowledge of their corresponding epitopes is of major interest. One possible approach for epitope characterization is the application of protein antigen proteolysis in combination with mass spectrometric peptide mapping analysis. Two complementary analytical strategies were applied: (a) limited proteolysis of antibody-bound antigen followed by removal of nonbound peptides and detachment of the antigenic peptides (epitope excision) and (b) enzymatic digest of the antigen followed by extraction of the antigenic peptides with the antibody and detachment of antigenic peptides after removal of nonbinding fragments (epitope extraction). In the few examples published so far, immobilized antibodies were used for these studies. In this study we present a method for characterization of the epitope sequences without prior immobilization of the monoclonal antibody. The separation of nonepitope peptides from antibody-bound peptides was carried out by ultrafiltration. The epitope and nonepitope fractions were analyzed by MALDI-MS without further purification, and the epitope sequences were identified. The method was developed using a model system consisting of the synthetic C-terminal cyanogen bromide fragment CB3 of myoglobin and the commercial monoclonal anti-myoglobin MG1. In further investigations the epitope sequence of a synthetic 32 amino acid peptide derived from heart muscle protein troponin T toward a monoclonal antibody MAb-M7, which was raised against the intact protein, was characterized. With this approach the epitope binding site of this antibody was determined, and selective shielding of potential cleavage sites in the immune complex could be observed. Furthermore, statements about the three-dimensional structure of the bound antigen were made.
Macht
M
M
University of Konstanz, Germany.
Fiedler
W
W
Kürzinger
K
K
Przybylski
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D015415:Biomarkers; D002417:Cattle; D002942:Circular Dichroism; D003488:Cyanogen Bromide / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009203:Myocardial Infarction / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry*; D009211:Myoglobin / Q000737:chemistry* / Q000276:immunology*; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014336:Troponin / Q000737:chemistry* / Q000276:immunology*; D020107:Troponin T
D000911:Antibodies, Monoclonal; D015415:Biomarkers; D000939:Epitopes; D009211:Myoglobin; D010446:Peptide Fragments; D014336:Troponin; D020107:Troponin T; D010450:Endopeptidases; D003488:Cyanogen Bromide