Micro-flow hydrophilic interaction liquid chromatography coupled with triple quadrupole mass spectrometry detects modified nucleosides in the transfer RNA pool of cyanobacteria
Micro-flow hydrophilic interaction liquid chromatography coupled with triple quadrupole mass spectrometry detects modified nucleosides in the transfer RNA pool of cyanobacteria
复制标题
微流亲水相互作用液相色谱结合三重四极杆质谱检测蓝藻转移 RNA 池中的修饰核苷
DOI：
10.1002/jssc.202100417
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发表时间：
2021-07-11
影响因子：
3.1
通讯作者：
Lin, Huan
中科院分区：
文献类型：
--
作者：
Qin, Yichao;Zhong, Qisheng;Lin, Huan
核苷的转录后修饰几乎存在于RNA的所有元件中。修饰的核苷可以微调RNA分子的结构，并影响生命功能，例如转移RNA的修饰摆动位置34，将反密码子的阅读偏好扩大到密码子。最近的研究表明，RNA元件中的修饰种类及其频率并不稳定，而是随着特定的细胞因素而变化，包括代谢物和特定的蛋白质(编写器、读取器和擦除器)。为了了解动态RNA修饰和生物过程之间的联系，需要灵敏和可靠的方法来确定修饰的核苷。本研究建立了微流控(8MU L/min)亲水作用液相色谱/三重四极杆联用技术同时测定腺苷、尿苷、胞苷、鸟苷和20种修饰核苷的方法。该方法用0.1-1000 nm标准(类似于0.03-300 ng/mL)进行了校正，并成功地应用于模型蓝藻细长聚球藻PCC7942中转移RNA修饰的测定。对分离清洁转移RNA池的方法进行了优化，仅需25 ng即可鉴定和定量转移RNA修饰。这种微流态液相色谱-串联质谱学方法是监测有限RNA样品中动态核糖核苷修饰的第一步。
Post-transcriptional modification of nucleosides is observed in almost all elements of RNA. Modified nucleosides finely tune the structure of RNA molecules and affect vital functions, such as the modified wobble position 34 of transfer RNAs expanding the reading preference of anticodons to codons. Recent investigations have revealed that the modification species and their frequencies in an RNA element are not stable but vary with specific cellular factors including metabolites and particular proteins (writers, readers, and erasers). To understand the link between dynamic RNA modifications and biological processes, sensitive and reliable methods for determining modified nucleosides are required. In this study, micro-flow (8 mu L/min) hydrophilic interaction liquid chromatography was coupled with triple quadrupole mass spectrometry for the simultaneous determination of adenosine, uridine, cytidine, guanosine, and 20 modified nucleosides. The method was calibrated using 0.1-1000 nM standards (similar to 0.03-300 ng/mL) and successfully applied to the determination of transfer RNA modifications in the model cyanobacterium Synechococcus elongatus PCC 7942. A protocol for the isolation of a clean transfer RNA pool was optimized, requiring only 25 ng for the identification and quantification of transfer RNA modifications. This micro-flow liquid chromatography-tandem mass spectrometry method constitutes the first step toward monitoring dynamic ribonucleoside modifications in a limited RNA sample.