雌二醇代謝物已被發現會與生物體脱氧核醣核酸反應形成脫嘌呤加合物，而這些反應也被認為與某些癌症的開端相關，然而其與體內物質形成鍵結的完整反應機構及位點仍是存有疑慮的。直到近期，才被發現雌二醇代謝物會與半胱胺酸、離胺酸、組胺酸形成共價性鍵結，且從糖尿病患者的血清中發現，雌二醇代謝物也會鍵結於人類血清蛋白、免疫球蛋白G上，以此正名，與雌激素代謝物產生共價性鍵結的蛋白即雌激素化蛋白。另外研究也指出，人類胰島素鍵結雌二醇代謝物會影響細胞內的信號傳送及糖吸收能力。
在此，我們開發且設計出一套利用銅催化疊氮基與炔類環化加成反應搭配生物素與鏈親和素之強鍵結力的雌激素化蛋白純化系統，以液相層析質譜裝置進行偵測。而在實驗參數的優化過程中，我們以人類胰島素作為模板蛋白，同時從全蛋白及蛋白胜肽層面觀察，順利提升了純化的效力。
隨後，我們在大鼠肝臟組織中添加已知雌激素化及非雌激素化蛋白來判斷複雜樣品純化效力。分別使用與活性物質兒茶酚雌激素反應之樣品；以及僅添加等量溶劑的樣品。經由純化後，再以雙甲基標定法分別標定兩組樣品各為氫及氘，最後以氘氫比值來判斷蛋白被雌激素化之準確性。於此我們純化出69個較具可信度及305個有雌激素化可能性的蛋白。最後我們利用了肝臟組織的催化效能，將添加於大鼠肝臟組織內的活性物質雌激素催化成活性物質兒茶酚雌激素，純化後進行雙甲基標定，一共鑑定出約312個有雌激素化可能的蛋白。
此純化系統除了擁有良好效能，在搭配雙甲基標定法後更能提升其準確性，因此我們也希望利用此系統偵測到更多的雌激素化蛋白，進而去了解其對於生理上的影響與重要性。

　　Quinoidal catechol estrogens (CE-Qs) are known to react with DNA and form stable adducts that are believed to be initiators of estrogen-induced tumorigenesis. Endogenous proteins that are covalently modified by catechol estrogens (CEs), referred to as estrogenized proteins, were recently identified from the blood serum of diabetic patients. In addition, insulin estrogenization was reported to affect insulin signaling and glucose uptake levels in cells. However, identification of estrogenized cellular proteins remains a challenging task.
Here, we developed a cleavable enrichment strategy via Cu-catalyzed azide-alkyne cycloaddition of an estrogen probe, 17α-Ethynylestradiol (EE2) or 4-Hydroxy Ethynyl Estradiol (4OHEE2), and biotin-based affinity enrichment for the identification of estrogenized proteins in cells or tissues using liquid chromatography-mass spectrometry (LC-MS). Target proteins in rat liver tissues were estrogenized by either 4OHEE2 probes upon co-incubation or EE2 probes by redox conversion. Estrogenized proteins were then clicked with a cleavable (disulfide linkage) biotin moiety, captured by avidin beads, and the captured (estrogenized) proteins were cleaved by the reduction of dithiothreitol followed by enzyme digestion. The resulting peptide digests were injected into LC-MSMS for protein identification based on proteomics approach. In order to differentiate non-specific capture by affinity beads, stable isotope dimethyl labeling was applied to tag the solution without (light) and with (heavy) the addition of the estrogen probe, respectively. Furthermore, human serum albumin, which is prone to estrogenization, and teriparatide (Forteo), which resists estrogenization, were spiked into the solution as the positive and negative control, respectively. Based on stringent search criteria and using the ratio (heavy/light) of the negative control as a filter, more than 300 proteins were identified from rat liver tissue to be potential targets of estrogenization. Among these target proteins, many are cytochrome enzymes that are involved in cellular metabolism or apoptosis and deserve further investigations.

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