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

摘要

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 α ligand-binding domain (hERα 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 hERα LBD. The relative gas-phase stability of selected hERα 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 17β-estradiol (E2) used as a reference ligand. Among the ligands tested, the relative binding affinity for hERα 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 (Kd) 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.