Proteomics Complementation of the Rat Uterotrophic Assay for Estrogenic Endocrine Disruptors: A Roadmap of Advancing High Resolution Mass Spectrometry-based Shotgun Survey to Targeted Biomarker Quantifications Authors: Laszlo Prokai *, Fatima Rahlouni, Khadiza Zaman,Vien Nguyen, and Katalin Prokai-Tatrai

The widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers uterine weight gain as endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomics by liquid chromatography–mass spectrometry on a high resolution (Orbitrap) instrument. Our samples were uterine protein extracts of ovariectomized rats after daily 17β-estradiol exposure for five days in comparison with those of vehicle-treated control animals. The study revealed that __ uterine proteins significantly regulated by estrogen treatment, and crucial findings were verified using multiple reaction monitoring-based targeted proteomics. When mapped by pathway analyses, estrogen-regulated proteins represented cell death and survival, cellular movement and protein synthesis as top molecular and cellular functions, and networks were found with the presence of nuclear estrogen receptor(s) as a prominent molecular node confirmed the relevance of our findings to hormone-associated events.