Estrogen receptor-ligand complexes measured by chip-based nanoelectrospray mass spectrometry: an approach for the screening of endocrine disruptors.
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.
Bovet
Cédric
C
Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland.
Wortmann
Arno
A
Eiler
Sylvia
S
Granger
Florence
F
Ruff
Marc
M
Gerrits
Bertran
B
Moras
Dino
D
Zenobi
Renato
R
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*
D052244:Endocrine Disruptors; D047628:Estrogen Receptor alpha; D008024:Ligands