Tatiana Pimenova1, Alexis Nazabal1, Bernd Roschitzki2, Jan Seebacher3,4, Oliver Rinner5, and Renato Zenobi1
- Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland
- Functional Genomics Center Zurich, UZH/ETH Zurich, CH-8057 Zurich, Switzerland
- Departments of Chemistry and Biochemistry, University of Washington, Seattle, Washington 98195, USA
- Institute for Systems Biology, Seattle, Washington 98103-8909, USA
- Institute for Molecular Systems Biology, ETH Zurich, CH-8093 Zurich, Switzerland
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.
CovalX Technology Used
DSS-d0/d12 and DSG-d0/d6 were used as reagents in the cross-linking experiments. 5 μL of a 6 μM solution of bPrP(25-241) was mixed with 5 μL of a 3 μM solutions of mAb3E7 before 1 μL of 3 mM solution of a 1:1 mixture of DSS-d0 and DSS-d12 or DSG-d0 and DSG-d6 in anhydrous DMF was added. Solutions of 10, 20, or 50-fold molar excess of reagent to total protein concentration were created. From this, it was found that the optimal molar excess was the 10-fold of excess reagent. Thus, the protein stock solutions were diluted with water accordingly. A control sample was prepared by repeating the process without adding the cross-linking reagent. All samples were incubated at room temperature and samples were taken at hour 1 and 2. The cross-linking reactions were quenched using ammonium bicarbonate solution to reach a final concentration of 4 mM. 1 μL of the protein solution was mixed with 1 μL of matrix (sinapic acid in 50% acetonitrile/0.1% TFA at a concentration of 10 mg/ml) in an Eppendorf tube before 1 μL of the final mixture was removed and spotted on a MALDI plate. The samples were analyzed using a mass spectrometer that had been modified with a CovalX HM1 detection system.