雌性荷爾蒙在生理體中的失衡常會造成許多疾病的發生，在文獻中也指出雌激素代謝物如醌化兒茶酚雌激素(CE-Qs)已被發現會與半胱胺酸、離胺酸、組胺酸形成共價性鍵結，而這些複合物也被認為是某些癌症的開端相關。雌激素代謝物與血清白蛋白形成雌激素化修飾蛋白質，利用標準添加法(Standard Addition Method)與多重反應監測模式(MRM/SRM transitions)的質譜分析方法達到鑑定以及定量血清中微量雌激素化修飾蛋白質。
　　經4-羥基雌二醇(4-OHE2)活化後的血清當作標準品。透過由上而下(Top Down)的完整蛋白分析，可以得到4-羥基雌二醇修飾在血清白蛋白上的數量；透過由下而上(Bottom Up)分析酵素水解後的胜肽，可以觀察到4-羥基雌二醇修飾在血清白蛋白上的位點。選擇三段分別含C58，K44，與K402修飾位點之胜肽發展多重反應監測模式經由參數設定可以在一次分析中，分析多個雌激素化修飾胜肽的前驅離子訊號，透過雌激素化修飾比率(Estrogenzied Ratio, ER)方程式，可以了解每段胜肽與雌激素共價鍵結的程度。
ER=((Intensity of modified ion))/[(Intensity of non-modified ion)+(Intensity of modified ion)] ×100%

　　微量的雌激素修飾蛋白無法精準的直接量測到，是因為濃度低於質譜儀的偵測極限，故透過標準添加法的方式，添加已知數量的標準品進入血清樣品中，可看出ER值隨著添加活化血清的量增加由約0.02%增加至2.32% ，呈現很好的線性增加關係，證明該修飾偵側訊號來自雌激素修飾。
　　此外，每一添加點的ER值三重複的標準偏差(RSD)含未添加點分別為44-76% (C58),24-64% (K44), 與39-73% (K402)，未添加點之RSD均落於平均值附近，且標準偏差值與平均值並無絕對關係。藉由線性回歸方程式的計算，回推出樣品中每段胜肽的內生性的雌激素化修飾比率與未添加之結果在RSD上亦無太大差異。顯示蛋白MRM方式的變異有待改進。但針對7位病人血清做定量，ER值在<100% RSD範圍內，對病人內生含量高低有一致性關係，約略是正常者的2~5倍。顯示本方法能用於血液內雌激素化修飾比率，經由定量血清樣品中的雌激素化修飾比率，有助於醫療上判斷雌性荷爾蒙失調造成的疾病的發病機制或是診斷依據。

Estrogens and their metabolites play key roles in many aspects of a woman’s health and diseases. Traditional methods used for estrogen quantification such as radioimmunoassay (RIAs) or electro chemiluminescence immuneassay (ECLIAs) have drawbacks in cross reactivity of antibodies used for the detection and fluctuation of hormone depending on the period of menstruation. In this study, a LC-MS based quantification method for catechol estrogens (CEs) in bloods was developed based on multiple reaction mode (MRM). Instead of free estrogen molecules, this method was established based on the detection of CEs-adducted serum proteins and proteomics techniques including sample preparation to yield digested peptides. Standard addition method was also applied to confirm the established method.
The collected serum samples were cleaned up by precipitation of trichloroacetic acid, digested by chymotrypsin. Three precursor peptides covering modification sites on C58 (sequence, m/z=778.54, z=3), K402 (sequence, m/z=1002.65, z=2), and K44 (sequence, m/z=632.71, z=2) of human serum albumin were chosen to develop the MRM transitions for quantification. Three product ions were chosen for each precursor ion. Moreover, three transitions for each non-modified peptide of the same sequence were also included in MRM method. This resulted in a total of 18 transitions (nine for modified and non-modified, respectively) in MRM method development. Estrogenized ratios were calculated as the ratio of sum of three transition intensity or area divided by the total sum of three transitions from both modified and non-modified precursors (equation). Standard addition method was further conducted by spiking different concentrations of in-vitro modified human serum albumin ranging from 0.02% to 2.32% into the serum samples. The data show good linearity (R2>0.95) with a relative standard deviation ranging from 27.0%- 81.4% based on three replicates. The method was then applied to quantify ER in seven collected serums.

The method was applied to detect ER in serums derived from one normal individual and six patients. The acquired ER values ranging were from 4.20E-05 - 2.35E-03 with RSD ranging from 12%-116%. The ER values of the patient are generally larger than that of the only normal one. A large scale quantification on more patients will be conducted based on the method developed in this study.

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