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-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.
Yu
L
L
Michael Barber Centre for Mass Spectrometry, Department of Chemistry, UMIST, Manchester, United Kingdom.
Gaskell
S J
SJ
Brookman
J L
JL
D000911:Antibodies, Monoclonal / Q000032:analysis*; D000941:Antigens / Q000032:analysis*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping / Q000295:instrumentation / Q000379:methods*; D007120:Immunochemistry; D010446:Peptide Fragments / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012441:Saccharomyces cerevisiae; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014780:Viruses / Q000737:chemistry
D000911:Antibodies, Monoclonal; D000941:Antigens; D010446:Peptide Fragments; D011506:Proteins