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 a ligand-binding domain (hERa 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 hERa LBD. The relative gasphase stability of selected hERa 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 17b-estradiol (E2) used as a reference ligand. Among the ligands tested, the relative binding affinity for hERa 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 affinityThe estrogen receptor (ER) belongs to the nuclear receptor (NR) superfamily of ligand-activated transcription regulators, which are involved in many processes such as growth, organ differentiation, and development of reproductive tissues. The ER, which comprises a DNA-binding domain (DBD) and a ligand-binding domain (LBD), activates the transcription of target genes in response to the binding of estrogens, a group of steroid compounds, to the LBD. Once bound by hormones, the ER undergoes a conformational change that facilitates dimerization and subsequent interactions with the specific DNA sequence (Kumar and Chambon 1988;Steinmetz et al. 2001). The large hydrophobic cavity of the ER LBD allows the binding of a wide variety of nonsteroidal compounds through hydrophobic interactions. In the last decades, a variety of Reprint requests to: Renato Zenobi, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland; e-mail: zenobi@org.chem.ethz.ch; fax: 41 44 632 12 92.Article published online ahead of print. Article and publication date are at

Probing the hydrophobic effect of noncovalent complexes by mass spectrometry

“…However, when the interaction in solution is determined by the entropy-driven hydrophobic effect, nanoESI-MS fails to give accurate results representative of the solution equilibria. Similar findings have been reported by other groups [16], including the study of hydrophobic ligands binding to protein targets [13][14][15]. If mixed polar/hydrophobic interactions are present (GST), the majority of the complex survives the transfer from solution into gas phase, most likely due to the polar fraction [16].…”

“…Nonetheless, there is some evidence that hydrophobic contributions do affect the gas-phase stability of complexes, although this is observed only in combination with electrostatic interactions [12]. Complexes that exist in solution mainly due to hydrophobic effects are likely to dissociate in the gas phase [11,[13][14][15][16]. When studying noncovalent complexes by ESI-MS, it is known that by increasing the ionic strength or adding organic solvents to the aqueous buffer will affect the hydrophobic effect in solution [17].…”

Protein Structural Studies by Traveling Wave Ion Mobility Spectrometry: A Critical Look at Electrospray Sources and Calibration Issues

“…Advantages of nanoESI include improved desolvation, greater salt tolerance, and higher ion yield [32,41,42]. However, nanoESI tends to be less robust in terms of signal stability and reproducibility than regular ESI [43,44]. It is often implied that nanoESI is better suited for preserving proteinligand interactions [32,45], although experimental evidence suggests that this is not necessarily the case [46][47][48].…”

Mass Spectrometric Characterization of HIV-1 Reverse Transcriptase Interactions with Non-nucleoside Reverse Transcriptase Inhibitors

“…[26][27][28] Several reports have shown that MS can be used to characterize the binding of drugs to target enzymes in terms of stoichiometry, specificity, and stability. 29,30) This method is advantageous because it requires small sample amounts. Because of its rapidity, sensitivity, and ability to directly determine binding stoichiometry, MS is potentially applicable as a superior high-throughput screening method for developing anti-HIV drugs.…”