文獻指出兒茶酚雌激素(Catechol Estrogen, CE)共價修飾是雌激素失衡和癌症的潛在生物標誌，而兒茶酚雌激素代謝物：高活性的醌類(quinone)致癌性物質，已被證實能夠與血清白蛋白上的離胺酸 (Lysine, K)、半胱胺酸 (Cysteine, C)和組胺酸(Histidine, H)進行共價鍵結形成雌激素修飾蛋白，由於蛋白具有較長的半生期，因此測量蛋白上的加成物更能反應出這些化學致癌物的水平。
本篇論文開發利用平行反應監測模式(PRM)來去偵測雌激素化蛋白，並定量血清白蛋白上兒茶酚雌激素的共價修飾程度，並應用在臨床樣品上評估其可行性。在此，我們證明了雌激素與HSA的共價修飾程度可以用來測定人類血液中循環兒茶酚雌激素水平。透過加入一系列不同濃度的兒茶酚雌激素與血清一起活化，產生各種共軛-CE/HSA比例，透過完整蛋白質的測量與分析，可以得到兩個線性良好的濃度範圍: 0-19μg/mL (R2=0.97)與57-1900 μg/mL (R2=0.99)；接著用胰凝乳蛋白酶與胰蛋白酶的消化，並利用nano-LC Q-Orbitrap平行反應模式(精確的MS2質量<0.05Da)靶向定量C34、K20、K378位點片段(胰凝乳蛋白酶)；K20、C34、K73、K281、H338、K378位點片段(胰蛋白酶)的修飾與未修飾片段。透過胰凝乳蛋白酶的消化，透過兩種共價程度位點之合併的計算(共價程度/Site、共價程度/HSA)，皆會得到良好的線性且RSD下降，但與完整蛋白斜率趨勢相反；而與胰凝乳蛋白酶相比，胰蛋白酶能夠得到更低的定量極限，且RSD較小(C34：2.96-28.90%)，與共軛-CE/HSA相比，在低濃度下數據趨近吻合(斜率=1.9)，主要為C34之貢獻，且R2>0.99。最後，我們將此方法應用在代謝異常病患與健康者的血清臨床樣品上，在完整蛋白分析，病患平均皆比健康者高，p值為0.000379，顯示兩者有顯著差異；使用胰蛋白酶部分，雖然經T.Test測試目前並無顯著差異，但病患平均可以看到比健康者高。我們證明了雌激素與HSA的共價修飾程度可以用來測定人類血液中循環兒茶酚雌激素水平。

Recent study points out that covalent modification of Catechol Estrogen (CE) is an estrogen imbalance and a potential biomarker for cancer. In this study, a method developed to detect and relative quantitative CEs-adducted serum proteins based on LC-MS/MS using parallel reaction monitoring (PRM). Instead of free estrogen molecules, the protein has a longer half-life, CEs-adducted serum proteins are more able to reflect these chemical carcinogen levels in blood.
Blood serum was added a series of concentrations of CEs to yield various conjugated-CE/HSA ratios’ serum standards. Through the measurement and analysis of intact proteins, exhibited two good linearity could be obtained: 0-19 μg/mL (R2=0.97) and 57-1900 μg/mL (R2=0.99). These serum standards were cleaned up by trichloroacetic acid (TCA) and digested by chymotrypsin and trypsin. In bottom-up analysis three conjugated and non-conjugated peptide pairs covering the site of K20、C34、K378 for chymotrypsin and six conjugated and non-conjugated peptide pairs covering the site of K20、C34、K73、K281、H338、K378 for trypsin were identified and targeted by PRM with accurate fragment mass (<0.05 Da), using nanoLC QE-Orbitrap. In the two- different enzymes system, the conjugation level/ site and conjugation level/ HSA derived from 18 fragments (chymotrypsin) and 63 fragments (trypsin) were revealed to have good linearity and RSD. However, the limit of quantification and the RSD calculated in the trypsin system were lower than it was in the chymotrypsin system.
Compared with the conjugated-CE/HSA, the data was close to the agreement at the low concentration (slope=1.9), mainly contribution was the site of C34 and had good linearity (R2=0.99). The method was applied to clinical samples to detect conjugation level in serum on 10 healthy individuals and 10 patients. In the intact protein analysis, patient conjugation level could see the significant difference (p=0.000379). In the bottom-up analysis, we can not see the significant difference between the T.Test but the average of patient can be seen to be higher than healthy.

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