Mass Spectrometric Mapping of Protein Epitope Structures of Myocardial Infarct Markers Myoglobin and Troponin T

Abstract

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.

Determination of primary structure and microheterogeneity of a β-amyloid plaque-specific antibody using high-performance LC–tandem mass spectrometry

“…In the case of the 6E10 heavy chain, only the variable region V H (1-50) spanning the CDR1 (residues 26-35) could be assigned by searching the MS/MS data against the NCBInr database. The fragment ion spectra (Supporting Information) for peptides containing the regions V H (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), V H (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) and V H (39-50) are consistent with the sequence indicated in Figure 1. According to the Kabat rules, the V H CDR1, with a typical length of 10-12 amino acids, is located four residues after the first cysteine of the variable region (CXXX) and is followed always by a tryptophane (20,21).…”

“…Among these, affinity-based approaches, in conjunction with high resolution and high sensitivity mass spectrometric methods, are becoming increasingly effective for identification of antigens directly from biological material (16). The developments in soft ionization techniques (17,18) and mass analyzers greatly expanded the potential of mass spectrometry to analyze large biomolecules, such as antibodies, and this has become a major tool for the characterization of recombinant protein pharmaceuticals (19).…”

Characterisation of the covalent structure of proteins from biological material by MALDI mass spectrometry ‣ possibilities and limitations

“…The study of enzyme specificity and kinetics [117], of protein interactions [6], of protein tertiary [118] and supramolecular structures [16], and of immune specificity [119,120] should be mentioned in this context. Based on the combination of 2D-PAGE protein separation with MALDI-MS analysis, the proteome [121] research as a complement to the genome projects has been started recently and, since then, dramatically accelerated.…”

In silico Epitope Mapping of Glucose-6-Phosphate Isomerase: A Rheumatoid Arthritis Autoantigen

“…Epitope mapping approaches can be broadly classified as either structural (X-ray crystallography, nuclear magnetic resonance, and electron microscopy) or functional (competition assays, antigen modification, antigen fragmentation and synthetic peptides) [18]. Many of the functional approaches are performed in conjunction with mass spectrometry (recently reviewed in Opuni et al [19]), and the most commonly used mass spectrometry-based epitope mapping approaches are epitope excision [20] and epitope extraction [21]. However, most of the experimental epitope mapping approaches are costly, time-consuming, and sometimes not viable since some antigens are insoluble in nondenaturing buffer solutions.…”