本研究運用高解析度質譜 (high-resolution mass-spectrometery, HRMS) 線性離子阱混成軌道阱質譜儀 (LTQ/Orbitrap-MS) 的資料依靠收集中性丟失啟動MS3掃描模式 (data-dependent neutral loss triggering MS3 scan, DD-NL-MS3) 建立基因鍵結體學 (DNA adductomics)。當MS1的母離子與MS2的子離子之間存在著五碳醣 (116.0474 amu) 的精確質量差，則進一步啟動MS3碎裂，以篩選分析所有DNA 鍵結物 (DNA adducts)，並獲得分子結構資訊。為了使DD-NL-MS3分析方法能有高敏感度以提升MS2及MS3啟動監測的機率，本研究利用經酵素水解的小牛胸腺DNA基質添加18種2''-deoxynucleosides混和標準品進行參數最佳化。優化MSn的離子儲存自動增益控制 (automatic gain control, AGC) 及離子注入時間 (ion injection time, IT)，最終參數設定為MSn AGC 50000及MSn IT 50 ms。 本研究運用最佳化後的LTQ/Orbitrap-MS DD-NL-MS3掃描模式來篩選並識別經甲醛暴露的去氧核苷 (2''-deoxynucleosides) 及小牛胸腺DNA所產生的DNA adducts全貌。並以LTQ/Orbitrap-MS數據為金標準，來評估先前本實驗在低解析度質譜-液相層析三段四極柱質譜儀 (liquid chromatography triple quadrupole mass spectrometer, LC-QqQ-MS) 已建立的中性丟失方法 (neutral loss) 搭配資料依靠收集模式 (information dependent acquisition) 觸發子離子掃描 (enhanced product ion) 篩檢DNA adducts的能力。本研究發現LC-QqQ-MS對DNA adducts的分子結構推測困難，但可作為初步篩檢工具來探討是否有DNA adducts因毒物暴露而形成。後續若能再搭配HRMS所帶來的高質量準確度及完整的結構斷片資訊 (MS2與MS3)，將使DNA adductomics更具優勢。 This study developed a DNA adductomics approach using a high-resolution mass-spectrometry (HRMS) linear ion trap hybrid orbitrap mass spectrometer (LTQ/Orbitrap-MS) with data-dependent neutral loss triggering MS3 scanning mode (DD-NL-MS3). With DD-NL-MS3 scan mode, the MS3 fragmentation event will be triggered if in the MS2 a neutral loss of one 2''-deoxyribose (dR; 116.0474 amu) from the parent ion was detected. The method was optimized using a representative sample of 18 modified 2''-deoxynucleosides spiked into calf thymus DNA hydrolysates. Parameters including MSn automatic gain control (AGC) and ion injection time (IT) were optimized, and were set as 50000 of AGC and 50 ms of IT. The developed HRMS approach was applied to detect the totality of formaldehyde-induced DNA adducts both in 2''-deoxynucleosides and calf thymus DNA. Furthermore, the results obtained from HRMS were used as the gold standard to evaluate the ability of broadly screening for DNA adducts using a low resolution MS [i.e., liquid chromatography triple quadrupole mass spectrometer (LC-QqQ-MS), operating in neutral loss scan mode triggered enhanced product ion (EPI) scan using information-dependent acquisition (IDA) criteria]. Our results show that DNA adductomics using LC-QqQ-MS alone could not provide sufficient information to elucidate the molecular structure of DNA adducts, but it still could be used as a preliminary screening tool to broadly detect the potential DNA adducts. To further combine the data of exact mass and MSn fragmentation profiles derived from HRMS, it will make DNA adductomics more advantageous.
