Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA.
Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
Rapid Commun Mass Spectrom. 2021 Jul 15;35(13):e9095. doi: 10.1002/rcm.9095.
As a new approach to DNA adductomics, we directly reacted intact, double-stranded (ds)-DNA under warm conditions with an alkylating mass tag followed by analysis by liquid chromatography/mass spectrometry. This method is based on the tendency of adducted nucleobases to locally disrupt the DNA structure (forming a "DNA bubble") potentially increasing exposure of their nucleophilic (including active hydrogen) sites for preferential alkylation. Also encouraging this strategy is that the scope of nucleotide excision repair is very broad, and this system primarily recognizes DNA bubbles.
A cationic xylyl (CAX) mass tag with limited nonpolarity was selected to increase the retention of polar adducts in reversed-phase high-performance liquid chromatography (HPLC) for more detectability while maintaining resolution. We thereby detected a diversity of DNA adducts (mostly polar) by the following sequence of steps: (1) react DNA at 45°C for 2 h under aqueous conditions with CAX-B (has a benzyl bromide functional group to label active hydrogen sites) in the presence of triethylamine; (2) remove residual reagents by precipitating and washing the DNA (a convenient step); (3) digest the DNA enzymatically to nucleotides and remove unlabeled nucleotides by nonpolar solid-phase extraction (also a convenient step); and (4) detect CAX-labeled, adducted nucleotides by LC/MS or a matrix-assisted laser desorption/ionization (MALDI)-MS technique.
Examples of the 42 DNA or RNA adducts detected, or tentatively so based on accurate mass and fragmentation data, are as follows: 8-oxo-dGMP, ethyl-dGMP, hydroxyethyl-dGMP (four isomers, all HPLC-resolved), uracil-glycol, apurinic/apyrimidinic sites, benzo[a]pyrene-dGMP, and, for the first time, benzoquinone-hydroxymethyl-dCMP. Importantly, these adducts are detected in a single procedure under a single set of conditions. Sensitivity, however, is only defined in a preliminary way, namely the latter adduct seems to be detected at a level of about 4 adducts in 10 nucleotides (S/N ~30).
CAX-Prelabeling is an emerging new technique for DNA adductomics, providing polar DNA adductomics in a practical way for the first time. Further study of the method is encouraged to better characterize and extend its performance, especially in scope and sensitivity.
作为 DNA 加合物组学的一种新方法，我们直接在温暖的条件下使完整的双链 (ds)-DNA 与烷基化质量标记物反应，然后通过液相色谱/质谱进行分析。该方法基于加合物碱基局部破坏 DNA 结构的趋势（形成“DNA 泡”），从而增加其亲核（包括活性氢）位点优先进行烷基化的暴露。也鼓励这种策略是核苷酸切除修复的范围非常广泛，并且该系统主要识别 DNA 泡。
选择具有有限非极性的阳离子二甲氧基苯（CAX）质量标记物，以增加极性加合物在反相高效液相色谱（HPLC）中的保留时间，从而提高检测的可检测性，同时保持分辨率。我们通过以下步骤序列检测到多种 DNA 加合物（主要是极性的）：（1）在存在三乙胺的情况下，在 45°C 下使 DNA 在水性条件下与 CAX-B（具有苄基溴官能团以标记活性氢位点）反应 2 小时；（2）通过沉淀和洗涤 DNA 去除残留试剂（方便的步骤）；（3）用酶消化 DNA 成核苷酸，并通过非极性固相萃取去除未标记的核苷酸（也是方便的步骤）；和（4）通过 LC/MS 或基质辅助激光解吸/电离（MALDI）-MS 技术检测 CAX 标记的加合核苷酸。
在所检测到的 42 个 DNA 或 RNA 加合物中，或根据准确质量和碎片数据推测为加合物的，有以下例子：8-oxo-dGMP、乙基-dGMP、羟乙基-dGMP（四种异构体，均在 HPLC 中分离）、尿嘧啶-乙二醇、无嘌呤/无嘧啶位点、苯并[a]芘-dGMP，以及首次检测到苯醌-羟甲基-dCMP。重要的是，这些加合物在单一条件下的单一程序中被检测到。然而，灵敏度仅以初步的方式定义，即后一种加合物似乎在约 10 个核苷酸中有 4 个加合物的水平被检测到（S/N≈30）。
CAX 预标记是 DNA 加合物组学的一种新兴新技术，首次以实用的方式提供了极性 DNA 加合物组学。鼓励进一步研究该方法以更好地表征和扩展其性能，特别是在范围和灵敏度方面。
