Department of Chemistry, University of Guanajuato, L de Retana No. 5, Guanajuato, Mexico.
Metallomics. 2010 Feb;2(2):140-6. doi: 10.1039/b915474d. Epub 2009 Dec 14.
Covalent modifications of nucleobases are thought to play an important role in regulating the functions of DNA and various cellular RNA types. Perhaps the best characterized is DNA methylation on cytosine (methyl tag attached to carbon 5 position) and such modification has also been detected in stable and long-lived RNA molecules. In this work, we propose a novel procedure enabling very sensitive quantification of methylcytidine and other ribonucleosides, based on reversed phase liquid chromatography with inductively coupled plasma mass spectrometry (ICP-MS) detection. The procedure relies on labeling ribose residues with osmium, by formation of a ternary complex between cis-diol ribose groups, hexavalent osmium (K(2)OsO(2)(OH)(4)) and tetramethylethylenediamine (TEMED). The derivatization reaction was carried out with 50 : 1 molar excess of Os to ribonucleoside, pH 4, for 2 h at room temperature. The structures of Os-labeled cytidine and methylcytidine were confirmed by electrospray ionization mass spectrometry. The separation of Os-labeled cytidine (C), uridine (U), 5-methylcytidine (5mC) and guanosine (G) was achieved on C18 column (Gemini, 150 × 3 mm, 5 μm) with isocratic elution (0.05% triethylamine + 6 mmol L(-1) ammonium acetate, pH 4.4: methanol (85 : 15)) and a total flow rate 0.6 mL min(-1). The column effluent was on-line introduced to ICP-MS (a model 7500 ce, Agilent Technologies) for specific detection at (189)Os. Calibration was performed within the concentration range 0-200 nmol L(-1) of each ribonucleoside and the analytical figures of merit were evaluated. For 100 μL injection, the detection limits for C, U, 5mC, G were 24, 38, 21 and 28 pmol L(-1), respectively. While introducing Os(vi)-TEMED to the column, it eluted in the dead volume and the detection limit for osmium was 20 pmol L(-1). The results obtained in this work might be helpful in the analysis of RNA digests, providing quantitative data on the ribonucleoside composition and RNA methylation (measured as the percentage of methylated cytidines with respect to total RNA cytidines).
碱基的共价修饰被认为在调节 DNA 和各种细胞 RNA 类型的功能方面起着重要作用。也许最具特征的是胞嘧啶的 DNA 甲基化（甲基标记附着在碳 5 位），这种修饰也已在稳定和长寿的 RNA 分子中检测到。在这项工作中，我们提出了一种新的程序，基于反相液相色谱与电感耦合等离子体质谱（ICP-MS）检测，能够非常灵敏地定量测定甲基胞嘧啶和其他核糖核苷。该程序依赖于用锇标记核糖，通过 cis-二醇核糖、六价锇（K(2)OsO(2)(OH)(4)）和四甲基乙二胺（TEMED）之间形成三元络合物来实现。衍生化反应在室温和 50:1 的锇与核糖核苷摩尔过量下进行 2 小时，pH 值为 4。通过电喷雾电离质谱法证实了 Os 标记胞嘧啶和甲基胞嘧啶的结构。Os 标记的胞嘧啶（C）、尿嘧啶（U）、5-甲基胞嘧啶（5mC）和鸟嘌呤（G）在 C18 柱（Gemini，150×3mm，5μm）上通过等度洗脱（0.05%三乙胺+6mmol L(-1) 乙酸铵，pH4.4：甲醇（85：15））和总流速 0.6mL min(-1)实现分离。柱流出物在线引入 ICP-MS（Agilent Technologies 公司的 7500 ce 型）进行特定检测，检测（189）Os。在每个核糖核苷的 0-200nmol L(-1) 浓度范围内进行校准，并评估分析的主要指标。对于 100μL 进样，C、U、5mC、G 的检测限分别为 24、38、21 和 28pmol L(-1)。当将 Os(vi)-TEMED 引入柱中时，它在死体积中洗脱，锇的检测限为 20pmol L(-1)。本工作的结果可能有助于 RNA 消化物的分析，提供有关核糖核苷组成和 RNA 甲基化（以相对于总 RNA 胞嘧啶的甲基化胞嘧啶百分比来衡量）的定量数据。
