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 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 (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.