Epitope mapping on bovine prion protein using chemical cross-linking and mass spectrometry.
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.
Pimenova
Tatiana
T
Department of Chemistry and Applied Biosciences, ETH Zürich, CH-8093 Zürich, Switzerland.
Nazabal
Alexis
A
Roschitzki
Bernd
B
Seebacher
Jan
J
Rinner
Oliver
O
Zenobi
Renato
R
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*
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011328:Prions; D011994:Recombinant Proteins