Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry & Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China.
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
Anal Chem. 2024 Jul 16;96(28):11366-11373. doi: 10.1021/acs.analchem.4c01446. Epub 2024 Jul 6.
The dynamic landscape of cellular nucleotides/nucleosides associated with RNA metabolism, particularly in diseases like cancer, has spurred intensive interest. Here, we report a robust stable isotope-diluted UHPLC-ESI-MS/MS method for accurate quantification of 12 purine ribonucleosides, including 10 methylated purine nucleosides. By the use of thermally decomposable ammonium bicarbonate (NHHCO) as a mobile phase additive for UHPLC-MS/MS detection, the ESI-MS/MS signal responses of these target compounds were enhanced by 1.7-24.5 folds. Noteworthily, three methylated guanosine isomers (m1G, m2G, and m7G) and two methylated adenosine isomers (m1A and m6A) that are indistinguishable directly by mass spectrometry were well resolved with optimal UHPLC separation. Combined with methanol extraction and solid-phase extraction (SPE) pretreatment, the method quantified intracellular concentrations of three modified nucleosides (Gm, m1G, and m2G), which would otherwise be undetectable because of significant suppression of their signals by the interfering cellular matrix. Nine purine nucleosides were simultaneously quantified in 293T cells, and their concentrations ranged by 4 orders of magnitude. Overall, the method presents high recovery rates over 90% for endogenous modified purine nucleosides in cultured cells, along with good precision, linearity, and LOD ranging from 0.30 fmol to 0.37 pmol per 5 × 10 cells. The developed UHPLC-MS/MS method holds potential for screening purine nucleosides as diagnostic and prognostic biomarkers and for quantifying purine epigenetic nucleosides post-cell metabolome analysis, thereby providing a valuable analytical tool for intracellular nucleoside quantification in future clinical research.
与 RNA 代谢相关的细胞核苷酸/核苷的动态景观，特别是在癌症等疾病中，引起了人们的浓厚兴趣。在这里，我们报告了一种稳健的稳定同位素稀释 UHPLC-ESI-MS/MS 方法，用于准确定量 12 种嘌呤核糖核苷，包括 10 种甲基化嘌呤核苷。通过使用热分解碳酸氢铵（NHHCO）作为 UHPLC-MS/MS 检测的流动相添加剂，这些目标化合物的 ESI-MS/MS 信号响应增强了 1.7-24.5 倍。值得注意的是，三种甲基鸟嘌呤异构体（m1G、m2G 和 m7G）和两种甲基腺苷异构体（m1A 和 m6A）通过质谱无法直接区分，通过最佳 UHPLC 分离得到了很好的分辨。结合甲醇提取和固相萃取（SPE）预处理，该方法定量了细胞内三种修饰核苷（Gm、m1G 和 m2G）的浓度，否则由于其信号受到细胞基质的显著抑制而无法检测到。在 293T 细胞中同时定量了 9 种嘌呤核苷，其浓度范围跨越 4 个数量级。总体而言，该方法在培养细胞中对内源性修饰嘌呤核苷的回收率超过 90%，具有良好的精密度、线性和 LOD，范围为 0.30 fmol 至 0.37 pmol/5×10 个细胞。开发的 UHPLC-MS/MS 方法具有作为诊断和预后生物标志物筛选嘌呤核苷的潜力，并可用于定量细胞代谢组分析后的嘌呤表观遗传核苷，从而为未来临床研究中细胞内核苷定量提供了有价值的分析工具。
