Quantitative Proteomic Investigation of Estrogenic Endocrine-Disrupting Effects in the Rat Uterus

The mammalian uterus is one of the most sensitive organs for estrogenicity. However, the widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers the uterine wet weight gain as an endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomic analysis by liquid chromatography–mass spectrometry from uterine protein extracts of ovariectomized rats after daily 17β-estradiol exposure for five days. We performed shotgun proteomic analysis of the uterus to identify candidate proteins for use as markers of estrogenicity. In addition, we mapped the differentially expressed proteins from untargeted analysis to signaling networks and biological processes through Ingenuity Pathway Analysis. We selected twelve of the top up- and down-regulated proteins for further evaluation by selected reaction monitoring-based targeted quantitation. Of the final six candidate markers, we verified all six as markers of estrogenicity by the application of the panel to testing rats exposed to a low and high dose of the known estrogenic compound bisphenol A. Altogether, the results of this study demonstrate the power of combining untargeted and targeted quantitative proteomic methods for a comprehensive analysis in rat uterus to evaluate changes in protein expression levels due to estrogen exposure, and to uncover candidate markers of estrogenicity in the development of a targeted proteomics panel.