Claudia Bich1, Mike Scott2, Andreas Panagiotidis3, and Ryan J. Wenzel1,4
- Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland
- Functional Genomics Center Zurich, CH-8057 Zurich, Switzerland
- Institute of Molecular Systems Biology (IMSB), ETH Zurich, CH-8093 Zurich, Switzerland
- CovalX, CH-8005, Zurich, Switzerland
The interaction between the bovine prion protein (bPrP) and a monoclonal antibody, 1E5, was studied with high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and surface plasmon resonance (SPR). In the case of MS a cross-linking stabilization was used prior to the analysis, whereas for SPR the antibody was immobilized and bPrP was injected. We compared the determination of parameters such as the epitope, the kinetics and binding strength, and the capacity of the antigen to bind two different antibodies. The two methods are highly complementary. SPR measurements require a lower amount of sample but are more time-consuming due to all of the necessary side steps (e.g., immobilization, regeneration). High-mass MALDI MS needs a higher overall amount of sample and cannot give direct access to the kinetic constants, but the analysis is faster and easier compared with SPR.
CovalX Technology Used
Using 1 μL of the CovalX K200 Stabilization Kit (30 to 50 μM) and 10 μl of sample solution (1 to 5 μM) were mixed and incubated at room temperature for 30 minutes or 2 hours. A matrix was created by dissolving 10 mg sinapic acid in 1 ml of water/acetonitrile/TFA (1:1:0.001). The cross-linked proteins were mixed in a 1:1 ratio with the matrix solution and 1 μL of the final mixture was spotted on a MALDI plate. The samples were then analyzed using a mass spectrometer that had been modified with a CovalX HM1 detection system. The data were background subtracted and smoothed using the CovalX Complex Tracker software.