title: Epitope mapping on bovine prion protein using chemical cross-linking and mass spectrometry.
abstract: An analytical strategy for the analysis of antigen epitopes by chemical cross-linking and mass spectrometry is demonstrated. The information of antigen peptides involved in the binding to an antibody can be obtained by monitoring the antigen peptides modified by a partially hydrolyzed cross-linker in the absence and in the presence of an antibody. This approach was shown to be efficient for characterization of the epitope on bovine prion protein bPrP(25-241) specifically recognized by a monoclonal antibody, 3E7 (mAb3E7), with only a small amount of sample (200 picomoles) needed. After cross-linking of the specific immuno complex, a matrix-assisted laser desorption/ionization (MALDI) mass spectrometer equipped with an ion conversion dynode (ICD) high-mass detector was used to optimize the amount of cross-linked complex formed at 202 kDa before proteolytic digestion. To identify the cross-linked peptides after proteolysis without ambiguity, isotope-labeled cross-linkers, disuccinimidyl suberate (DSS-d0/d12) and disuccinimidyl glutarate (DSG-d0/d6), together with high-resolution Fourier transform ion-cyclotron resonance mass spectrometry (FTICR-MS) were used. As a result, a complete fading of the peak intensities corresponding to the peptides representing the epitope was observed when bPrP/mAb3E7 complexes were formed.
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zürich, CH-8093 Zürich, Switzerland.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Pimenova
authors.forename: Alexis
authors.initials: A
authors.lastname: Nazabal
authors.forename: Bernd
authors.initials: B
authors.lastname: Roschitzki
authors.forename: Jan
authors.initials: J
authors.lastname: Seebacher
authors.forename: Oliver
authors.initials: O
authors.lastname: Rinner
authors.forename: Renato
authors.initials: R
authors.lastname: Zenobi
mesh_terms: D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry*; D018604:Epitope Mapping / Q000379:methods*; D036103:Nanotechnology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010449:Peptide Mapping; D011328:Prions / Q000737:chemistry* / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011328:Prions; D011994:Recombinant Proteins