RAPID LABEL-FREE QUANTITATIVE PROTEOMICS OF ESTROGENIC ENDOCRINE-DISRUPTING EFFECTS IN THE RAT UTERUS USING A SYSTEMS BIOLOGY APPROACH

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2013-04-12

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Sahyouni, Fatima

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Purpose: To identify estrogen-induced differential protein expression in rat uterine tissue using a rapid label-free proteomics and systems biology approach. Methods: Ovariectomized rats were treated short-term with subcutaneous E2 injections using corn oil as a vehicle. Approximately 10 mg of tissue were dissected from the uterus of vehicle-treated control and E2-treated animals for proteomic analyses. Uterine proteins were extracted with 8M urea for 30 minutes and subsequently processed by reduction, alkylation and digestion for mass spectrometry analysis. The samples were analyzed using a hybrid linear ion trap-Fourier transform ion cyclotron mass spectrometer equipped with an electrospray ionization source and connected to a nanoflow liquid chromatography system. MS/MS data was searched against a composite IPI rat protein database containing both forward and randomized sequences using the Mascot software. Quantitation was performed using an MS/MS-based total ion currents (TICs) approach using the Scaffold software. Additionally, Ingenuity Pathway Analysis (IPA) was utilized to derive interaction networks among the identified proteins. Results: The mammalian uterus increases its weight due to fluid imbibition and cell proliferation by exogenously administered estrogenic compounds. With the observation of weight gain in the treated uterus compared to non-treated control rats, we confirmed E2's uterotrophic effects for our subsequent proteomics study. Out of a total of 262 identified proteins, 163 proteins were differentially regulated (with p<0.05 considered statistically significant) by the hormone. Of the 163 proteins that were significantly regulated, 153 were up-regulated in E2-treated uteri and 10 were down-regulated in E2-treated uteri. These 163 proteins were submitted and mapped into 19 networks that merged into E2-regulated pathways. Top networks included molecular transport, carbohydrate metabolism, cancer, developmental disorders and cellular function and maintenance. Implicated diseases were endocrine system and metabolic disorders. Top signaling pathways involved metabolic pathways, steroid signaling and actin cytoskeleton signaling. Conclusions: In addition to the expected increase in wet uterine weights, we have elucidated and organized a large number of E2-induced protein expression changes into interaction networks. Metabolism and developmental disorders were implicated as the top networks. (Supported by the Robert A. Welch Foundation, BK-0031, and the NIH grant AG031535)

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