EPITOPE MAPPING OF MONOCLONAL-ANTIBODIES BY MASS-SPECTROMETRY - IDENTIFICATION OF PROTEIN ANTIGENS IN COMPLEX BIOLOGICAL-SYSTEMS

We describe the application of immunoaffinity extraction and mass spec trometry to the analysis of Ty1 Gag protein in lysates of Saccharomyce s cerevisiae. A magnetic bead-conjugated monoclonal antibody was used to achieve selective extraction, the specificity of which was establis hed by matrix-assisted laser desorption/ionization mass spectrometric (MS) analysis of an extract of the lysate of cells overexpressing the Ty1 Gag protein. MS analysis of similar extracts of lysates following tryptic hydrolysis confirmed selective extraction of the epitope-conta ining peptide fragment. Sufficient sensitivity was achieved to allow t he application of this approach to the analysis of lysates of wild-typ e cells. Furthermore, the sequence of the epitope-containing peptide w as confirmed by electrospray-tandem MS. To our knowledge, this constit utes the first report of the application of immunoaffinity extraction and tandem MS analysis to the characterization of an antigen recovered from a complex cellular system. (C) 1998 American Society for Mass Sp ectrometry.