Cédric Bovet1, Benoit Plet2, Marc Ruff3, Sylvia Eiler3, Florence Granger3, Andreas Panagiotidis4, Ryan Wenzel2, Alexis Nazabal2, Dino Moras3, and Renato Zenobi1
- Department of Chemistry and Applied Biosciences, ETH Zurich, HCI E 329, 8093 Zurich, Switzerland
- CovalX AG, Technoparkstrasse 1, 8005 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
- Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland
High-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking has the ability to monitor the ligand-dependent dimerization of the human estrogen receptor α ligand binding domain (hERα LBD) in solution. Because only ER ligands enhance the homodimer abundance, we evaluated the ability of this label-free approach for identifying endocrine disrupting compounds (EDCs) in a high-throughput manner. This was achieved by combining an automated liquid handler with an automated MS acquisition procedure, which allowed a five-fold gain in operator time compared to a fully manual approach. To detect ligand binding with enough confidence, the receptor has to be incubated with at least a 10 μM concentration of the test compound. Based on the increase of the measured homodimer intensity, eight compounds with a relative binding affinity (RBA, relative to the natural hormone estradiol) >7% were identified as ER ligands among the 28 chemicals tested. Two other compounds, quercetin and 4-tert-amylphenol, were also identified as ER ligands, although their RBAs have been reported to be only 0.01% and 0.000055%, respectively. This suggests that these two ligands have a higher affinity for hERα LBD than reported in the literature. The high-mass MALDI approach thus allows identifying high affinity EDCs in an efficient way.
hERα LBD stock solution (2.8 mg/ml in 10% glycerol, 100 mM NaCl, 100 mM KnaHPO4, pH 7.4) was diluted 16 times in deionized water (pH 7.4) before 5 μL of the solution was incubated with 5 μL of ligand dissolved in deionized water for 15 minutes. 1 μL of cross-linking solution was used to stabilize the protein complexes and the cross-linking reactions occurred using a 4 mg/ml solution of the CovalX K200 Stabilization Kit. The mixture was incubated at room temperature for 60 minutes before 1 μL was removed and mixed with 1 μL of matrix (sinapic acid (10 mg/ml) in acetonitrile:water (1:1, v/v) acidified with 0.1% TFA). 1 μL of the final mixture was dropped onto a MALDI sample 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.