基于质谱的RNA及其修饰分析

doi:10.16288/j.yczz.25-052

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Hereditas(Beijing) ›› 2025, Vol. 47 ›› Issue (8): 885-902.doi: 10.16288/j.yczz.25-052

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Mass spectrometry-based analysis of RNA and its modifications

Ying Feng(), Xiaoli He(), Yu Liu(), Jin Wang()

State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot 010020, China

Received:2025-02-20 Revised:2025-06-18 Online:2025-06-19 Published:2025-06-19
Contact: Yu Liu, Jin Wang E-mail:1650977020@qq.com;xiaolihe2022@163.com;jinwang@imu.edu.cn;liuyu_j@126.com
Supported by:
National Natural Science Foundation of China(32360147);Natural Science Foundation of Inner Mongolia Autonomous Region(2022QN02013);2022 High-Level Returned Overseas Talents Program]

Abstract

Abstract:

Ribonucleic acids (RNAs) are key biomolecules responsible for the transmission of genetic information, the synthesis of proteins and its regulation, and modulation of many biochemical processes. They are also the key components of many viruses. Chemically modified synthetic RNAs or oligoribonucleotides are becoming more widely used as therapeutics and vaccines. Demands for technologies to detect, sequence, identify, and quantify RNA and its modifications far exceed requirements found in the DNA realm. Currently, mass spectrometry (MS) has become the primary technology for identifying, sequencing, and quantifying RNA and its modifications. This paper mainly reviews latest advances in mass spectrometry for the research of RNA and its modifications, and discusses the strengths and weaknesses of this technology, aiming to provide readers with a comprehensive perspective from technical fundamentals to application prospects, promote the broader application of mass spectrometry in RNA research, and provide important references for method developers and biological researchers in the field.

Key words: RNA modification, post-transcriptional modification, epitranscriptome, mass spectrometry, LC-MS/MS

Ying Feng, Xiaoli He, Yu Liu, Jin Wang. Mass spectrometry-based analysis of RNA and its modifications[J]. Hereditas(Beijing), 2025, 47(8): 885-902.

Figures/Tables 6

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Table 1

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酶	切割特异性	局限性	参考文献
RNase A	嘧啶核苷和假尿苷的3′端	会生成较短的降解产物，不利于修饰位点的确定	[100]
hRNase 4	位于嘌呤5′端且与之相邻的尿苷	适用于mRNA和其他长链RNA，但需要添加T4多核苷酸激酶以避免生成2′,3′-环磷酸酯	[101]
RNase T1	鸟苷和N²-甲基鸟苷(m²G)的3′端	无法生成高的序列覆盖度，尤其是在存在富含G的序列冗余时	[102]
MC1	尿苷和假尿苷的5′端	不能区分假尿苷和尿苷	[87]
Cusativin	胞苷和m⁵C的5′端	连续胞苷残基之间没有磷酸二酯键切割	[103]
RNase H	DNA-RNA杂合链中的RNA	可能产生非特异性切割	[104~106]
RNase U2	嘌呤残基处切割RNA	生成2′,3′-环磷酸酯和3′-线性磷酸酯消化产物，使得质谱分析变得复杂	[107]
colicin E5	在某些tRNA的第34位的辫苷(Q)与第35位的尿苷之间切割	仅对大肠杆菌的特定tRNA具有切割作用	[108,109]
mazF	在5′-NAC-3′序列(N优先为U或A)中的N与A之间切割	对RNA底物的识别和切割活性易受RNA二级结构的影响	[110]

软件	优点	缺点	参考文献
NIST spectral software	可鉴定特定修饰的存在或不存在	不能区分尿苷和胞苷；只能用于已知的寡核苷酸	[128]
RAMM	计算机(in silico)数据库；固定和可变测序	不能区分相同经典核苷的甲基化修饰和硫修饰；不能完全消除对数据的人工解析	[125,126]
RoboOligo	复杂修饰和多个修饰的从头测序	不能区分核苷位置异构体；不能区分m/z值相同或几乎相同的母离子；不能区分尿苷和假尿苷	[129]
NASE	开源软件；校正母离子质量及阳离子加合；基于目标/诱饵(target/decoy)搜索策略的错误发现率(FDR)参数	高错误发现率；不能完全消除对数据的人工解析；不能区分核苷位置异构体；不能区分尿苷和假尿苷	[124]
Pytheas	开源软件；适用于同位素标记序列；基于目标/诱饵搜索策略的FDR 能够区分位置异构体；能够区分尿苷和假尿苷	需要人工解析数据；需要大量计算	[123]
Ariadne	RNA鉴定和测序自动化	缺乏通过FDR计算对匹配输出结果进行验证；不能区分核苷位置异构体；不能区分尿苷和假尿苷	[127,130]
Nucleo-SAFARI	计算机(in silico)数据库；自动注释在轨道阱质谱仪平台上记录的核酸top-down MS/MS谱图；基于R语言的核酸MS/MS谱图分析；直接基于m/z识别碎片，而无需依赖去卷积和去同位素算法；能够区分位置异构体	不能对多个质谱图和序列进行批量处理	[131]

Mass spectrometry-based analysis of RNA and its modifications

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