兒茶酚雌激素 (Catechol Estrogen, CE) 是雌激素的代謝物之一，其醌類 (Quinone) 化合物為具高活性的致癌物質。近期研究發現，雌激素於人類血清白蛋白上的修飾會影響其抗氧化及運輸物質的功能。而組蛋白轉譯後修飾會影響染色質的反應，包括轉錄、基因靜默及其穩定性，已有許多文獻指出組蛋白上的修飾失衡與疾病或癌症有所關聯，故透過研究兒茶酚雌激素修飾蛋白，可幫助我們了解疾病發生機制或開發生物指標。

透過螢光的測定，我們首次發現兒茶酚雌激素化蛋白在500 nm激發波長下具有紅螢光特性，並可藉由溶液及凝膠 (SDS-PAGE) 螢光來直接看出兒茶酚雌激素化蛋白的存在。而透過完整蛋白質譜分析，我們發現兒茶酚雌激素修飾在H1上的訊號，並計算兒茶酚雌激素在人類血清白蛋白與組蛋白上的修飾比例。在同濃度兒茶酚雌激素下組蛋白的凝膠螢光訊號雖然與人類血清白蛋白相像，但其兒茶酚雌激素修飾比例卻遠低於人類血清白蛋白。

另一方面，轉譯後修飾的鑑定常常會遇到一些困難，如樣品量不足、生物樣品的複雜性以及需高靈敏度的分析技術。為了克服這些困難，我們使用了富集策略、分離技術以及高解析度與靈敏度質譜儀，以提升轉譯後修飾鑑定的可能性。透過鍵擊化學親和性純化法將兒茶酚雌激素化人類血清白蛋白純化富集後，於凝膠上能看出明顯的螢光訊號。而在完整蛋白的質譜分析上，也成功看到兒茶酚雌激素化人類血清白蛋白被純化富集出的訊號，證實此鍵擊化學親和性純化富集方法確實可純化出兒茶酚雌激素化目標蛋白。此結果有助於對蛋白轉譯後修飾相關資訊的研究。唯兒茶酚雌激素化組蛋白的部分，雖然於凝膠上可看出強烈的螢光訊號，表示鍵擊化學親和性純化法確實能純化出兒茶酚雌激素化組蛋白，卻無法從質譜上看出其修飾訊號，在此推測為離子抑制造成的影響。

Catechol estrogen (CE) is one of the metabolites of estrogen, and its quinone forms are highly active carcinogens. Recent studies have found that the modification of estrogens on human serum albumin (HSA) affects its antioxidant and transportation functions. Post-translational modifications of histones can affect chromatin responses, including transcription, gene silencing and their stability. Much research has pointed out that the modification imbalance on histones is associated with diseases or cancers. Therefore, by studying CE-adducted proteins, we are able to further understand the mechanism of disease development and to develop biomarkers.

For the first time we found that the CE-adducted proteins have red fluorescence characteristics at the excitation wavelength of 500 nm, and the adducts can be directly identified by solution and gel (SDS-PAGE) exposed under UV. By intact protein analysis, the signal of CE modification on H1 was successfully identified, and the adduction ratio of CE on HSA and histone was calculated. We found that the fluorescence signal of histones on the gel exposed under UV was similar to that of HSA when both proteins were incubated with identical amount of CE, although the CE adduction ratio of histone is much lower than that of HSA.

On the other hand, identification of post-translational modifications (PTMs) often encounters difficulties such as insufficient sample size, complexity of biological samples, and analytical techniques requiring high sensitivity. To overcome these difficulties, enrichment strategies, separation techniques, and high-resolution and sensitive mass spectrometry were utilized to improve the identification of PTMs. After click chemistry-based affinity enrichment of CE-HSA adducts, the significant fluorescence signal of the adducts on the gel, as well as the signals of those on mass spectra, were successfully observed, confirming the feasibility of the enrichment strategy we developed. These results are helpful in our protein PTMs-related studies. However, even though the fluorescence of CE-histone adducts on the gel was clear and obvious, the signals of them on mass spectra were not the case, which we suspect was the result of ion suppression.

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