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Estrogen receptor–ligand complexes measured by chip-based nanoelectrospray mass spectrometry: An approach for the screening of endocrine disruptors

¹ Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland
² Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS, 67404 Illkirch, France
³ Functional Genomics Center, UZH | ETH Zurich, 8057 Zurich, Switzerland

(RECEIVED November 15, 2006; FINAL REVISION January 22, 2007; ACCEPTED February 12, 2007)

In the present report, a method based on chip-based nanoelectrospray mass spectrometry (nanoESI-MS) is described to detect noncovalent ligand binding to the human estrogen receptor ligand-binding domain (hER LBD). This system represents an important environmental interest, because a wide variety of molecules, known as endocrine disruptors, can bind to the estrogen receptor (ER) and induce adverse health effects in wildlife and humans. Using proper experimental conditions, the nanoESI-MS approach allowed for the detection of specific ligand interactions with hER LBD. The relative gas-phase stability of selected hER LBD–ligand complexes did not mirror the binding affinity in solution, a result that demonstrates the prominent role of hydrophobic contacts for stabilizing ER–ligand complexes in solution. The best approach to evaluate relative solution-binding affinity by nanoESI-MS was to perform competitive binding experiments with 17-estradiol (E2) used as a reference ligand. Among the ligands tested, the relative binding affinity for hER LBD measured by nanoESI-MS was 4-hydroxtamoxifen diethylstilbestrol > E2 >> genistein >> bisphenol A, consistent with the order of the binding affinities in solution. The limited reproducibility of the bound to free protein ratio measured by nanoESI-MS for this system only allowed the binding constants (K_d) to be estimated (low nanomolar range for E2). The specificity of nanoESI-MS combined with its speed (1 min/ligand), low sample consumption (90 pmol protein/ligand), and its sensitivity for ligand (30 ng/mL) demonstrates that this technique is a promising method for screening suspected endocrine disrupting compounds and to qualitatively evaluate their binding affinity.

Keywords: electrospray ionization mass spectrometry; noncovalent; nuclear receptor; estrogen receptor; endocrine disruptors; solution affinity

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