運用連線固相萃取-液相層析三段式四極柱串聯質譜儀發展基因鍵結體學

本研究利用連線固相萃取 (on-line SPE) 液相層析三段式四極柱串聯質譜儀 (LC-QqQ-MS) 建立基因鍵結體學 (DNA adductomics) 分析法。採用質譜技術的中性丟失模式 (constant neutral loss, CNL)， 以 2’-deoxyribose 作為分析特徵以大範圍量測 adducted 2’-deoxynucleosides。使用18種去氧核糖核苷混合標準品進行參數最佳化，包括電噴灑離子化的溫度 (heater temperature)、去簇電壓 (declustering potential, DP) 以及碰撞能量 (collision energy, CE) 提高了分析的敏感度。最終參數的設定為 : 電噴灑離子化溫度 400 °C 、DP 30伏特及CE 10-30伏特。 本研究將建立的方法運用至體外及體內暴露甲基化試劑的DNA 樣本，以全面量測 DNA 鍵結物。CNL模式下分析的數據經輸出並繪製成基因鍵結體地圖 (adductome map)，可完整呈現 DNA 損傷樣貌，並比對樣本間的訊號差異。此外，本研究在 (multiple reaction monitoring) 模式下搭配已知標準品平行定量暴露甲基化試劑的 DNA 樣本，驗證所開發的基因鍵結體學分析法。最後，為了推測目標訊號的分子結構，本研究嘗試以中性丟失方法 (CNL) 搭配資料依靠收集模式 (information dependent acquisition) 運用子離子掃描 (enhanced product ion) 取得目標訊號的斷片資訊。

We developed a DNA adductomics methodology using liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS). By using the constant neutral loss (CNL) scanning mode, we are able to comprehensively detect the DNA adducts resulting from the CNL loss of 2’-deoxyribose. We firstly optimized the parameters of electrospray ionization (ESI)-QqQ-MS using a 18 nucleosides standard mixture solution. These parameters included ESI temperature, declustering potential (DP) and collision energy (CE). The optimized parameters are found to be: ESI temperature set at 400 °C, DP at 30 volts and CE at a range of 10-30 volts. This DNA adductomic method is then applied in DNA samples measurement obtained from in vitro and in vivo experiments, in which the DNA samples were pre-treated with methylating agents. The obtained data were transferred and output to establish an adductome map to comprehensively show the DNA damages. Furthermore, in order to verify the DNA adductomic method, we also applied the quantitation method (using multiple reaction monitoring mode) to quantify the DNA adducts induced by the methylating agents. Finally, in order to further identify the chemical structure of target analyte, we further employed an information dependent acquisition (IDA) method to trigger enhanced product ion (EPI) scan that can give information about the fragmentation pattern of unidentified nucleosides.