RIBONUCLEOSIDE ANALYSIS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY
RIBONUCLEOSIDE ANALYSIS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY
复制标题
DOI：
10.1016/s0021-9673(00)94152-9
复制
发表时间：
1989-06-02
期刊：
影响因子：
--
通讯作者：
KUO, KC
中科院分区：
文献类型：
--
作者：
GEHRKE, CW;KUO, KC
在过去的十五年中，我们开发和完善了使用反相高效液相色谱和紫外光电二极管阵列检测（RPLC-UV）检测和测量核酸和生物液体中主要和修饰核苷的分析色谱方法。RPLC-UV核苷分析，因为它现在已经发展成为一个强大的新的研究工具，以帮助研究人员在生物化学和生物医学研究领域。这种RPLC-UV核苷方法可以在一次分析中分离超过65个核苷，“运行间”峰保留变化小于1%。一个完整的核苷组成可以从少至0.5 μg RNA获得。从几个皮摩尔（约1 ng）的核苷的保留时间和特征紫外光谱可以鉴定和确认核苷，本文介绍了用于分析RNA中核苷和核苷组成的标准RPLC-UV方法。介绍了色谱方案和标准核苷柱，并描述了HPLC仪器的基本要求。三个优化的RPLC系统的开发，特别强调的分辨率，速度或灵敏度。此外，选择三种未分级的tRNA作为参考核苷的来源，用于评估色谱的性能。从这些tRNA中，表征了用于该方法的标准化和校准的大量核苷。本文还讨论了采用二极管阵列检测器提高核苷鉴定的可靠性和测量的准确性。还描述了用于释放tRNA中的外来修饰的核苷的扩展的酶水解方案。大量核糖核苷的色谱保留时间和紫外光谱列表。RPLC-UV核糖核苷分析方案能够定量31个核苷。定量分析需要约1 μg的同功tRNA或20 μg的未分级tRNA。在此量的tRNA下，四种主要核苷的测量相对误差百分比小于2%，对于修饰的核苷约为5%。可分析的纯同受体tRNA仅为0.2 μg，但在如此低的样品量下，预计修饰核苷的相对误差为20-30%。对于rRNA中修饰核苷的定量，其修饰比tRNA少得多，每次进样需要10-100 μg样品。该量在常规分析柱（25 cm × 4.6 mm硅胶C18柱）的上样容量范围内。然而，在进样此量时，需要注意确保四种主要核苷的响应在检测器和数据简化系统的线性范围内。给出了16 S和23 SrRNA分析的定量数据，并举例说明了这种核苷方法在生物化学和生物医学研究中的一些独特而有趣的应用。
Over the past fifteen years we have developed and refined the analytical chromatographic methodologies using reversed-phase high-performance liquid chromatography and UV—photodiode array detection (RPLC—UV) for the detection and measurement of the major and modified nucleosides in nucleic acids and biological fluids. RPLC—UV nucleoside analysis as it has now evolved is a powerful new research tool to aid investigators in the fields of biochemical and biomedical research. This RPLC—UV nucleoside method can resolve more than 65 nucleosides in a single analysis with “run-to-run” peak retention variations of less than 1%. A complete nucleoside composition can be obtained from as little as 0.5 μg RNA. Identification and confirmation of nucleosides can be made from the highly reproducible retention times and from the characteristic UV spectrum from a few picomoles (ca.1 ng) of nucleoside.In this paper we introduce standard RPLC—UV methodologies for the analysis of nucleosides and nucleoside composition of RNAs. The chromatographic protocols and standard nucleoside columns are presented and the essential requirements necessary in the HPLC instrumentation are described. Three optimized RPLC systems were developed, each with particular emphasis placed on resolution, speed, or sensitivity. In addition, three unfractionated tRNAs were selected as sources of reference nucleosides are for assessment of the performance of the chromatography. From these tRNAs, a large array of nucleosides were characterized which are used in standardization and calibration of the method. Also discussed is the use of a diode-array detector for enhancement of the reliability of nucleoside identification and accuracy of measurement. An extended enzymatic hydrolysis protocol for the liberation of exotically modified nucleosides in tRNAs is also described. Chromatographic retention times and UV spectra for a large number of ribonucleosides are tabulated.The RPLC—UV ribonucleoside analytical protocols are capable of quantifying 31 nucleosides. Approximately 1 μg of an isoaccepting tRNA, or 20 μg of unfractioned tRNA are needed for quantitative analysis. With this amount of tRNA, the percent relative error of measurement of the four major nucleosides is less than 2%, and for the modified nucleosides about 5%. As little as 0.2 μg of pure isoaccepting tRNA can be analyzed, but at the expense of precision as at this low sample size a 20–30% relative error for modified nucleosides is to be expected.For quantitation of the modified nucleosides in rRNA, which contains much less modification than tRNAs, 10–100 μg of sample are needed per injection. This amount is within the loading capacity of a regular analytical column (25 cm × 4.6 mm silica based C18column). However, with this quantity injected, caution is required to ensure that the response for the four major nucleosides is within the linear range of the detector and data reduction system. Quantitative data from the analysis of 16S and 23S rRNA are given.Examples are presented of some unique and interesting applications of this nucleoside methodology to biochemical and biomedical investigations.