title: Estrogen receptor-ligand complexes measured by chip-based nanoelectrospray mass spectrometry: an approach for the screening of endocrine disruptors.
abstract: 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 alpha ligand-binding domain (hERalpha 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 hERalpha LBD. The relative gas-phase stability of selected hERalpha 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 17beta-estradiol (E2) used as a reference ligand. Among the ligands tested, the relative binding affinity for hERalpha LBD measured by nanoESI-MS was 4-hydroxtamoxifen approximately 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.
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland.
authors.forename: Cédric
authors.initials: C
authors.lastname: Bovet
authors.forename: Arno
authors.initials: A
authors.lastname: Wortmann
authors.forename: Sylvia
authors.initials: S
authors.lastname: Eiler
authors.forename: Florence
authors.initials: F
authors.lastname: Granger
authors.forename: Marc
authors.initials: M
authors.lastname: Ruff
authors.forename: Bertran
authors.initials: B
authors.lastname: Gerrits
authors.forename: Dino
authors.initials: D
authors.lastname: Moras
authors.forename: Renato
authors.initials: R
authors.lastname: Zenobi
mesh_terms: D052244:Endocrine Disruptors / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D047628:Estrogen Receptor alpha / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D036103:Nanotechnology / Q000379:methods; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D052244:Endocrine Disruptors; D047628:Estrogen Receptor alpha; D008024:Ligands