Institute of Chemistry, Eötvös Loránd University, Pázmány Péter stny. 1/A, H-1117 Budapest, Hungary.
MS Metabolomics Research Group, Centre for Structural Study, Research Centre for Natural Sciences, Eötvös Loránd Research Network, Magyar tudósok krt. 2, H-1117 Budapest, Hungary.
Anal Methods. 2021 Oct 14;13(39):4614-4622. doi: 10.1039/d1ay00897h.
Global DNA methylation and hydroxymethylation play an important role in gene expression. They can be connected with several diseases. The modification status could be a biomarker to determine the status of disease. A fast, easy and accurate liquid chromatography - tandem mass spectrometry method has been developed for the precise quantitation of 5-methylcytosine and 5-hydroxymethylcytosine. Formic acid was used for the hydrolysis of the DNA strand resulting in nucleobases. These polar hydrolysis products were separated on a normal phase column using reversed phase eluents in inverse gradient mode. Multiple reaction monitoring was applied to achieve high selectivity and sensitivity for the quantitation. A new relative quantitation model was developed by using guanine, as an internal standard, present in samples. The new method was successfully validated with excellent accuracy and precision values in the range of 0.005-0.5% for 5hmC and 1-15% for 5mC. The main advantages of this quantitation method are that, due to relative quantitation, calibration curves can be used without reacquiring the calibration points and no additional isotope labeled internal standards are required. The method was tested to identify the concentrations of 5mC and 5hmC in various sample types. The lowest level of DNA sample required in the case of 0.005% 5hmC is 0.5 μg.
全球 DNA 甲基化和羟甲基化在基因表达中起着重要作用。它们可以与几种疾病有关。修饰状态可以作为一种生物标志物来确定疾病的状态。已经开发出一种快速、简单、准确的液相色谱-串联质谱法，用于精确定量 5-甲基胞嘧啶和 5-羟甲基胞嘧啶。甲酸用于 DNA 链的水解，导致核苷碱。这些极性水解产物在正相柱上用反相洗脱剂在逆梯度模式下分离。采用多反应监测实现定量的高选择性和灵敏度。通过使用样品中存在的鸟嘌呤作为内标，开发了一种新的相对定量模型。新方法在 5hmC 的 0.005-0.5%和 5mC 的 1-15%范围内具有优异的准确性和精密度。该定量方法的主要优点是，由于相对定量，无需重新获取校准点即可使用校准曲线，并且不需要额外的同位素标记内标。该方法已用于检测各种样品类型中 5mC 和 5hmC 的浓度。在 0.005%5hmC 的情况下，所需的 DNA 样品的最低水平为 0.5 μg。
