Taniguchi H, Hayashi N
Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan.
Nucleic Acids Res. 1998 Mar 15;26(6):1481-6. doi: 10.1093/nar/26.6.1481.
Liquid chromatography/electrospray mass spectrometry is one of the rapidly developing techniques with which mass of large hydrophilic polymers such as proteins and nucleic acids can be determined precisely. The technique was applied to studies on the modifications of tRNAs. Various tRNA species purified from Escherichia coli were directly injected into a capillary reversed-phase column and the desalted and concentrated tRNAs were analyzed on-line with an electrospray mass spectrometer. In some cases, small but significant differences were noted between the theoretical and observed molecular masses, suggesting that there exist still unknown modifications. Under high resolution measurements, multiple peaks corresponding to species modified to a varying extent were resolved. To study the structures in detail, the isolated tRNA species were digested with ribonuclease T1, and the resulting mixture of fragments were analyzed by the same liquid chromatography/mass spectrometry. In this way, most of the fragments were easily identified solely from their masses, and the positions where the expected and real structures differ were revealed. The results obtained showed the presence of micro-heterogeneity among tRNAs and demonstrated at the same time the power of the hyphenated technique for the structural analysis on nucleic acids.
液相色谱/电喷雾质谱法是一种快速发展的技术，利用该技术可以精确测定蛋白质和核酸等大型亲水性聚合物的质量。该技术被应用于对转运核糖核酸（tRNA）修饰的研究。从大肠杆菌中纯化得到的各种tRNA种类被直接注入毛细管反相柱，然后用电喷雾质谱仪对脱盐和浓缩后的tRNA进行在线分析。在某些情况下，理论分子量和观察到的分子量之间存在微小但显著的差异，这表明仍存在未知的修饰。在高分辨率测量下，对应于不同程度修饰物种的多个峰被分辨出来。为了详细研究结构，将分离出的tRNA种类用核糖核酸酶T1消化，然后用相同的液相色谱/质谱法分析所得的片段混合物。通过这种方式，大多数片段仅根据其质量就很容易被鉴定出来，并且揭示了预期结构和实际结构不同的位置。所获得的结果表明tRNA之间存在微观异质性，同时也证明了联用技术在核酸结构分析方面的强大功能。
