Monitoring Ligand Modulation of Protein-Protein Interactions by Mass Spectrometry: Estrogen Receptor r-SRC1



Cédric Bovet1, Marc Ruff2, Sylvia Eiler2, Florence Granger2, Ryan Wenzel3, Alexis Nazabal3, Dino Moras2, and Renato Zenobi1


  1. Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland, IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire)
  2. Département de Biologie et Génomique Structurales, Université Louis Pasteur, U596 INSERM, UMR7104 CNRS, 1 rue Laurent Fries, 67404 Illkirch, France,
  3. 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

Complex Tracker


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.

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Categories : Publications, High-Mass MS