A simplified method for rapid quantification of intracellular nucleoside triphosphates by one-dimensional thin-layer chromatography
A simplified method for rapid quantification of intracellular nucleoside triphosphates by one-dimensional thin-layer chromatography
复制标题
DOI：
10.1016/j.ab.2010.10.029
复制
发表时间：
2011-02-15
影响因子：
2.9
通讯作者：
Martinussen, Jan
中科院分区：
文献类型：
--
作者：
Jendresen, Christian Bille;Kilstrup, Mogens;Martinussen, Jan
核苷酸定量是代谢组学的重要组成部分，但由于缺乏快速、灵敏和可靠的方法而受到阻碍。我们提出了一种更省时、更灵敏、更精确的方法来定量测定细胞提取物中的核苷三磷酸 (NTP)、5-核糖基-1-焦磷酸 (PRPP) 和无机焦磷酸 (PPi)。该方法使用一维薄层色谱 (TLC) 和放射性标记的生物样品。在优化的酸性甲酸铵和氯化物溶剂中以离子电荷水平解析核苷酸，从而可以对 NTP 进行定量。该方法比当前的二维方法和基于高效液相色谱 (HPLC) 的程序明显更简单、更快速，可实现更高的通量，同时避免了常见的不准确来源和技术问题。为了测定 PPi，用无机焦磷酸酶 (PPase) 处理放射性标记的磷酸盐，以去除污染的焦磷酸盐。一式三份进行的生物学实例显示，所确定的 NTP 浓度的标准偏差约为平均值的 10%。 (C) 2010 Elsevier Inc. 保留所有权利。
Quantification of nucleotides is an important part of metabolomics but has been hampered by the lack of fast, sensitive, and reliable methods. We present a less time-consuming, more sensitive, and more precise method for the quantitative determination of nucleoside triphosphates (NTPs), 5-ribosyl-1-pyrophosphate (PRPP), and inorganic pyrophosphate (PPi) in cell extracts. The method uses one-dimensional thin-layer chromatography (TLC) and radiolabeled biological samples. Nucleotides are resolved at the level of ionic charge in an optimized acidic ammonium formate and chloride solvent, permitting quantification of NTPs. The method is significantly simpler and faster than both current two-dimensional methods and high-performance liquid chromatography (HPLC)-based procedures, allowing a higher throughput while common sources of inaccuracies and technical problems are avoided. For determination of PPi, treatment with inorganic pyrophosphatase (PPase) of the radiolabeled phosphate is employed for removal of contaminating pyrophosphate. Biological examples performed in triplicates showed standard deviations of approximately 10% of the mean for the determined concentrations of NTPs. (C) 2010 Elsevier Inc. All rights reserved.