Cédric Bovet1, Marc Ruff2, Sylvia Eiler2, Florence Granger2, Ryan Wenzel3, Alexis Nazabal3, Dino Moras2, and Renato Zenobi1
- Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland, IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire)
- Département de Biologie et Génomique Structurales, Université Louis Pasteur, U596 INSERM, UMR7104 CNRS, 1 rue Laurent Fries, 67404 Illkirch, France,
- CovalX AG, Technoparkstrasse 1, 8005 Zurich, Switzerland
Many drugs and chemicals exert their biological effect by modulating protein−protein interactions. In vitro approaches to characterize these mechanisms are often based on indirect measurements (e.g., fluorescence). Here, we used mass spectrometry (MS) to directly monitor the effect of small-molecule ligands on the binding of a coactivator peptide (SRC1) by the human estrogen receptor α ligand binding domain (hERα LBD). Nanoelectrospray mass spectrometry (nanoESI-MS) and high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking were employed to follow these processes. The chemical cross-linking protocol used prior to high-mass MALDI analysis allows detection of intact noncovalent complexes. The binding of intact hERα LBD homodimer with two coactivator peptides was detected with nanoESI-MS and high-mass MALDI-MS only in the presence of an agonist ligand. Furthermore, high-mass MALDI-MS revealed an increase of the homodimer abundance after incubating the receptor with a ligand, independent of the ligand character (i.e., agonist, antagonist). The binding characteristics of the compounds tested by MS correlate very well with their biological activity reported by cell-based assays. High-mass MALDI appears to be an efficient and simple tool for directly monitoring ligand regulation mechanisms involved in protein−protein interactions. Furthermore, the combination of both MS methods allows identifying and characterizing endocrine-disrupting compounds or new drug compounds in an efficient way.
CovalX Technology Used
Protein stock solution was desalted and incubated with 1 μM of ligand for 15 minutes at room temperature. 1 μM of CAP was added and the mixture was incubated for 15 more minutes. The solution was then diluted to 5 μM using water. The protein complexes were stabilized using 1 μL of cross-linking solution (CovalX K200 Stabilization Kit) with 9 μL of proteins. The subsequent mixture was incubated or 60 minutes at room temperature and then 1 μL was removed. The removed sample was mixed with 1 μL of matrix (sinapic acid (10 mg/ml) in acetonitrile/water (1:1) acidified with 0.1% TFA). 1 μL of this final mixture was dropped on a MALDI sample plate and 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.