Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, Japan.
J Biosci Bioeng. 2013 Mar;115(3):332-8. doi: 10.1016/j.jbiosc.2012.10.006. Epub 2012 Nov 30.
Liquid chromatography/mass spectrometry is widely used for studying sequence determination and modification analysis of small RNAs. However, the efficiency of liquid chromatography-based separation of intact small RNA species is insufficient, since the physiochemical properties among small RNAs are very similar. In this study, we focused on ion mobility-mass spectrometry (IM-MS), which is a gas-phase separation technique coupled with mass spectrometry; we have evaluated the utility of IM-MS for microRNA (miRNA) analysis. A multiply charged deprotonated ion derived from an 18-24-nt-long miRNA was formed by electrospray ionization, and then the time, called the "drift time", taken by each ion to migrate through a buffer gas was measured. Each multivalent ion was temporally separated on the basis of the charge state and structural formation; 3 types of unique mass-mobility correlation patterns (i.e., chainlike-form, hairpin-form, and dimer-form) were present on the two-dimensional mobility-mass spectrum. Moreover, we found that the ion size (sequence length) and the secondary structures of the small RNAs strongly contributed to the IM-MS-based separation, although solvent conditions such as pH had no effect. Therefore, sequence isomers could also be discerned by the selection of each specific charged ion, i.e., the 6(-) charged ion reflected a majority among chainlike-, hairpin-, and other structures. We concluded that the IM-MS provides additional capability for separation; thus, this analytical method will be a powerful tool for comprehensive small RNA analysis.
液相色谱/质谱联用广泛用于研究小 RNA 的序列测定和修饰分析。然而，由于小 RNA 之间的理化性质非常相似，基于液相色谱的完整小 RNA 种类的分离效率不足。在本研究中，我们专注于离子淌度-质谱（IM-MS），这是一种与质谱联用的气相分离技术；我们评估了 IM-MS 用于 microRNA (miRNA) 分析的实用性。电喷雾电离产生源自 18-24nt 长 miRNA 的带多个电荷的去质子化离子，然后测量每个离子通过缓冲气体迁移所需的时间，称为“漂移时间”。基于电荷状态和结构形成，每个多价离子在时间上被分离；在二维淌度-质谱上存在 3 种独特的质量-淌度相关模式（即链式、发夹式和二聚体式）。此外，我们发现尽管溶剂条件（如 pH 值）没有影响，但小 RNA 的离子大小（序列长度）和二级结构强烈促成了基于 IM-MS 的分离。因此，还可以通过选择每个特定的带电离子来辨别序列异构体，即 6(-) 电荷离子反映了链式、发夹式和其他结构中的大多数。我们得出结论，IM-MS 提供了额外的分离能力；因此，这种分析方法将成为全面的小 RNA 分析的有力工具。
