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 spectrometry to the analysis of Ty1 Gag protein in lysates of Saccharomyces cerevisiae. A magnetic bead-conjugated monoclonal antibody was used to achieve selective extraction, the specificity of which was established by matrix-assist...

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Bibliographic Details
Published in:Journal of the American Society for Mass Spectrometry 1998-03, Vol.9 (3), p.208-215
Main Authors: Lu, Yu, Gaskell, Simon J., Brookman, Jayne L.
Format: Article
Language:English
Subjects:
Antibodies, Monoclonal - analysis
Antigens
Antigens - analysis
Baking yeast
Biological and medical sciences
Blotting, Western
Chromatography, Affinity
Desorption
Electrophoresis, Polyacrylamide Gel
Epitope Mapping - instrumentation
Epitope Mapping - methods
Extraction
Fundamental and applied biological sciences. Psychology
Genic rearrangement. Recombination. Transposable element
Immunochemistry
Ionization
Ions
Mass spectrometry
Molecular and cellular biology
Molecular genetics
Monoclonal antibodies
Peptide Fragments - analysis
Proteins
Proteins - analysis
Saccharomyces cerevisiae
Scientific imaging
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Spectroscopy
Viruses - chemistry
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Summary:We describe the application of immunoaffinity extraction and mass spectrometry to the analysis of Ty1 Gag protein in lysates of Saccharomyces cerevisiae. A magnetic bead-conjugated monoclonal antibody was used to achieve selective extraction, the specificity of which was established 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-containing peptide fragment. Sufficient sensitivity was achieved to allow the application of this approach to the analysis of lysates of wild-type cells. Furthermore, the sequence of the epitope-containing peptide was confirmed by electrospray-tandem MS. To our knowledge, this constitutes 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.
ISSN:1044-0305
1879-1123
DOI:10.1016/S1044-0305(97)00250-X