Towards High-Throughput Identification of Endocrine Disrupting Compounds with Mass Spectrometry



Cédric Bovet1, Benoit Plet2, Marc Ruff3, Sylvia Eiler3, Florence Granger3, Andreas Panagiotidis4, Ryan Wenzel2, Alexis Nazabal2, Dino Moras3, and Renato Zenobi1


  1. Department of Chemistry and Applied Biosciences, ETH Zurich, HCI E 329, 8093 Zurich, Switzerland
  2. CovalX AG, Technoparkstrasse 1, 8005 Zurich, Switzerland
  3. 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
  4. 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.



Complex Tracker


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.




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