雌二醇代謝物中的4OHE2 (4-hydroxyestradiol)，其醌類代謝物E2-3,4-Q為強的親電子劑，會修飾在DNA上並誘導癌症發生，而肥胖也會造成體內雌激素含量增加，進而導致癌症生成。測定蛋白質上雌激素修飾的比例，可以反應出人體在長時間下暴露於雌激素的程度，而血紅蛋白(Hb)在血液中是一種含量極為豐富的蛋白質，再加上在血液中有很長的壽命(約120天)，因此研究血紅蛋白與兒茶酚雌激素的共價修飾程度極其重要。
本實驗中透過SDS-PAGE觀察在不同4OHE2濃度影響下誘導血紅蛋白聚集的程度，可以發現當使用1.2 ug以上劑量的4OHE2與200 ug血紅蛋白反應3小時後，血紅蛋白dimer的量會明顯增加，且拖尾的情況也明顯出現。為了觀察血紅蛋白在正常情況下被4OHE2修飾的位點，且不希望蛋白聚集而影響到位點的偵測或誤判，所以透過SDS-PAGE實驗的觀察，我們決定標準品的反應條件為1 ug 4OHE2與200 ug Hb於37℃反應3小時。添加一系列不同濃度的4OHE2與血紅蛋白反應，在完整蛋白質的偵測(Intact Protein Measurement)下，可以看到4OHE2鍵結在Hb上的比例(Conjugated-CE/Hb)隨著4OHE2的濃度增加而有線性的成長，且線性關係極好(R² = 0.9994)。
使用胰凝乳蛋白酶水解標準品，並透過DDA (Data Dependent Acquisition)模式進行分析，偵測到β鏈上的K120與C93兩個位點有4OHE2修飾，之後透過PRM (Parallel Reaction Monitoring)模式進行偵測，並計算各位點在各濃度4OHE2下的共價修飾程度(Conjugation level)，做成檢量線後發現K120不具線性關係，且檢量線幾乎呈水平狀態，我們推測此修飾可能為分子量極為靠近4OHE2的其他化合物；C93的Conjugation level則有隨著4OHE2的濃度增加而顯著上升，且也具有好的線性關係(R² = 0.9574)，可以確定C93確實是會被4OHE2修飾的位點，另外在C93各濃度的Conjugation level與完整蛋白質測量中測得之Conjugated-CE/Hb做成的關係圖中也可以看到兩個數值具有極高的相關性(R² = 0.9544)。
此外比較了肥胖者與正常者之間C93的Conjugation level差異性。比較圖中可以看到正常者的平均值略高於肥胖者，其p- value為0.810，無顯著差異，但相對標準偏差都很大，除了紅血球本身為複雜樣品，會影響偵測外，低於LOQ也會使測得的數值較為浮動，另外也可能是因為第一重複與第二重複上機時，儀器的狀況有些差異，或是胰凝乳蛋白酶因專一性較差，導致水解蛋白時效率不好等因素所造成，需再進行第三重複樣品測定來確認其相對標準偏差。
此方法仍有可改進之處，但希望此方法的開發，能對肥胖或代謝症候群的人提供醫療診斷或評估上的幫助，並期望此研究能對醫療領域有所貢獻。

The catechol estrogen metabolite of 4OHE2 (4-hydroxyestradiol), E2-3,4-Q, is a strong electrophile, and it can conjugate with DNA and cause cancer. Hemoglobin (Hb) is a good target protein to study because of its high abundance in blood and long lifetime (about 120 days). Measuring the conjugation level of catechol estrogen in circulating blood with hemoglobin can reflect the level that the human body was exposed to 4OHE2 in a long time, so it is important to research the conjugation level of catechol estrogen with hemoglobin. In this experiment, the degree of hemoglobin aggregation which was induced by different concentrations of 4OHE2 was observed by SDS-PAGE. We could see that the amount of dimer increased significantly after incubation at 37 ℃ for 3 hours with 1.2 ug of 4OHE2 and 200 ug of hemoglobin, so we decided the incubation conditions of standard as below:1 ug of 4OHE2 and 200 ug of hemoglobin, 37 ℃ for 3 hours. In intact protein measurement, we could see Conjugated-CE/Hb increased linearly with the concentration addition of 4OHE2 (R² = 0.9994). In the bottom-up analysis, we found K120 and C93 of the β chain could be modified by 4OHE2, but only the conjugation level of C93 could increase linearly with the concentration addition of 4OHE2 (R² = 0.9574), so C93 was a modified site of 4OHE2. Moreover, the Conjugated-CE/Hb and conjugation level of C93 was also found they had a good correlation. Besides, we also compared C93 between obese and normal people, and the average value of the normal people was slightly higher than that of the obese people, and the p-value was 0.810. There was no significant difference, but the relative standard deviation was very large, maybe was due to the sample preparation, enzyme digestion or condition of instrument between two repeats. This method still needed to be improved, I hoped this method can contribute to the medical field.

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