在生物檢測上血清算是相當容易取得，而根據文獻研究指出可以在血清中發現雌激素代謝物如醌化兒茶酚雌激素(CE-Qs)等與血清白蛋白形成雌激素化修飾蛋白質，為了偵測血清中微量雌激素化修飾蛋白質，本實驗室建立一套結合固相萃取法與多重反應監測模式的質譜分析方法。在本研究中先進行多重反應監測模式離子對(MRM/SRM transitions)的選擇，MRM/SRM transitions的選擇為一個胜肽序列至少選擇三個序列片段進行偵測。在條件測試上所選用之素材為經4-羥基雌二醇(4-OHE2)活化後的血清白蛋白標準品，將所得的質譜數據經資料庫分析結果與本實驗室先前所發表文獻進行比對後，選定有重複比對到之胜肽序列作為母離子，再以母離子之二次質譜圖中前三強訊號為子離子，以此為建立多重反應監測模式之依據。為了使固相萃取達到較好的分離效果，故使用經4-羥基雌二醇活化後的血清樣品進行條件測試，在最終沖提條件下達到目標胜肽分離及去除雜質之目的。
此外，本實驗室將此分析方法用來檢測代謝異常之肥胖症患者與健康控制組的雌激素化修飾蛋白質的修飾比例，並將兩組的分析結果進行t-test統計分析得到0.05的p value，但雌激素化修飾蛋白質之修飾比例在兩個組別間仍具有2倍之差距。目前本實驗室嘗試利用此分析方法進行雌激素化修飾蛋白質的絕對定量，以期此方法能達到定量及定性之成效。

Recent studies have revealed that serum proteins form covalent bonds with active metabolites of estradiol, catechol estrogens in human blood. In this study, we developed a sensitive method for qualitative and quantitative detection of trace amount of estrogenized proteins in human blood by combining solid-phase extraction (SPE), bottom-up proteomics, and multiple reaction monitoring (MRM) of mass spectrometry (MS). For this development, purified human serum albumin (HSA) was activated by 4-hydroxyestradiol to form an estrogenized protein standard for method optimization. Based on previous data, the tryptic peptide that contains the estrogenized site at the only free cysteine residue Cys34 of HSA (ALVLIAFAQYLQQCPFEDHVK) was chosen as the target of the precursor ion and three product ions which cover the modification site was chosen to form three transitions, 906.47/1104.53, 906.47/1161.07, 906.47/1069.01. Solid-phase extraction (SPE) using reversed phase C18 cartridge helped to clean up the tryptic digest of the serum sample but significant sample lose was noticed. After optimization, wash solvent composed of 20% acetonitrile and elution solvent composed of 80% acetonitrile in 0.05% formic acid were used for SPE and the eluted fraction was injected onto Nano LC-MS/MS for analysis. The percentage of estrogenization was calculated based on the peak area of the modified ion divided by the sum of modified and non-modified ion of the same backbone. We applied this method to analyze the estrogenization percentage of HSA derived from 30 diabetic obese patients against those derived from 30 healthy control. Results indicated that the estrogenization percentage of the patient group was twice greater than that of the normal control with significant difference (p = 0.05).

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