Isotope-dilution mass spectrometry for exact quantification of noncanonical DNA nucleosides
Isotope-dilution mass spectrometry for exact quantification of noncanonical DNA nucleosides
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DOI：
10.1038/s41596-018-0094-6
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发表时间：
2019-01-01
期刊：
影响因子：
14.8
通讯作者：
Carell, Thomas
中科院分区：
文献类型：
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作者：
Traube, Franziska R.;Schiffers, Sarah;Carell, Thomas
DNA不仅含有典型的核苷酸，而且含有各种碱基的修饰。特别是胞嘧啶和腺嘌呤经常被修饰。确定这些非正则基的确切数量可以有助于表征生物系统的状态，例如，决定疾病或发育过程，因此非常重要。在这里，我们提出了一个工作流程，其中包括关键的样品制备步骤和质谱分析和验证的重要方面的详细描述。在这个协议中，提取和消化的DNA通过优化的旋转柱和酶为基础的方法进行了描述。在DNA消化过程中加入同位素标记的标准品，这允许通过同位素稀释质谱法进行精确定量。为了克服这种分析的主要瓶颈，我们开发了一种短（类似于14分钟每个样品）超高效液相色谱（UHPLC）和三重四极杆质谱（QQQ-MS）方法。使用所提供的评估表可以轻松计算基因组中的修饰丰度。与替代方法相比，这里提出的定量程序允许快速，超灵敏（低飞摩尔范围）和高度可重复的定量不同的核苷平行。包括样品制备和评估，DNA修饰的定量可以在不到一周的时间内实现。
DNA contains not only canonical nucleotides but also a variety of modifications of the bases. In particular, cytosine and adenine are frequently modified. Determination of the exact quantity of these noncanonical bases can contribute to the characterization of the state of a biological system, e.g., determination of disease or developmental processes, and is therefore extremely important. Here, we present a workflow that includes detailed description of critical sample preparation steps and important aspects of mass spectrometry analysis and validation. In this protocol, extraction and digestion of DNA by an optimized spin-column and enzyme-based method are described. Isotopically labeled standards are added in the course of DNA digestion, which allows exact quantification by isotope dilution mass spectrometry. To overcome the major bottleneck of such analyses, we developed a short (similar to 14-min-per-sample) ultra-HPLC (UHPLC) and triple quadrupole mass spectrometric (QQQ-MS) method. Easy calculation of the modification abundance in the genome is possible with the provided evaluation sheets. Compared to alternative methods, the quantification procedure presented here allows rapid, ultrasensitive (low femtomole range) and highly reproducible quantification of different nucleosides in parallel. Including sample preparation and evaluation, quantification of DNA modifications can be achieved in less than a week.