Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry.
Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry.
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MALDI-TOF质谱法鉴定酶降解产物中的RNA片段
DOI：
10.3791/63720
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发表时间：
2022-04-11
影响因子：
1.2
通讯作者：
Resendiz, Marino J. E.
中科院分区：
文献类型：
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作者：
Schowe, Shawn W.;Langeberg, Conner J.;Chapman, Erich G.;Brown, Kitty;Resendiz, Marino J. E.
RNA 是存在于生命各个领域的生物聚合物，其与其他分子和/或活性物质（例如 DNA、蛋白质、离子、药物和自由基）的相互作用无处不在。因此，RNA 会经历各种反应，包括裂解、降解或修饰，从而产生具有不同功能和影响的生物学相关物种。一个例子是鸟嘌呤氧化成 7,8-二氢-8-氧代鸟嘌呤 (8-oxoG)，这可能在活性氧 (ROS) 存在的情况下发生。总体而言，表征此类产品和转化的程序对科学界非常有价值。为此，基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱是一种广泛使用的方法。本协议描述了如何表征酶处理后形成的 RNA 片段。所选模型使用 RNA 和核糖核酸外切酶 Xrn-1 之间的反应，其中酶消化在氧化位点停止。通过固相合成获得两个20个核苷酸长的RNA序列[5'-CAU GAA ACA A(8-oxoG)G CUA AAA GU]和[5'-CAU GAA ACA A(8-oxoG)(8-oxoG) CUA AAA GU]，通过紫外-可见光谱进行定量，并通过MALDI-TOF进行表征。然后将获得的链进行（1）5'-磷酸化并通过MALDI-TOF进行表征； (2)用Xrn-1处理； (3)过滤、脱盐； (4)通过MALDITOF分析。该实验设置明确鉴定了与 Xrn-1 停滞相关的片段：[5'-H2PO4-(8-oxoG)G CUA AAA GU]、[5'-H2PO4-(8-oxoG)(8-oxoG) CUA AAA GU] 和 [5'-H2PO4-(8-oxoG) CUA AAA GU]。所述实验使用 200 皮摩尔 RNA（20 pmol 用于 MALDI 分析）进行；然而，较低的量可能会导致使用比本工作中使用的功率更大的激光源的光谱仪检测到峰值。重要的是，所描述的方法可以推广并有可能扩展到涉及 RNA 和 DNA 的其他过程的产品鉴定，并且可能有助于其他生化途径的表征/阐明。
RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, and free radicals, are ubiquitous. As a result, RNA undergoes various reactions that include its cleavage, degradation, or modification, leading to biologically relevant species with distinct functions and implications. One example is the oxidation of guanine to 7,8-dihydro-8-oxoguanine (8-oxoG), which may occur in the presence of reactive oxygen species (ROS). Overall, procedures that characterize such products and transformations are largely valuable to the scientific community. To this end, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is a widely used method. The present protocol describes how to characterize RNA fragments formed after enzymatic treatment. The chosen model uses a reaction between RNA and the exoribonuclease Xrn-1, where enzymatic digestion is halted at oxidized sites. Two 20-nucleotide long RNA sequences [5′-CAU GAA ACA A(8-oxoG)G CUA AAA GU] and [5′-CAU GAA ACA A(8-oxoG)(8-oxoG) CUA AAA GU] were obtained via solid-phase synthesis, quantified by UV-vis spectroscopy, and characterized via MALDI-TOF. The obtained strands were then (1) 5′-phosphorylated and characterized via MALDI-TOF; (2) treated with Xrn-1; (3) filtered and desalted; (4) analyzed via MALDITOF. This experimental setup led to the unequivocal identification of the fragments associated with the stalling of Xrn-1: [5′-H2PO4-(8-oxoG)G CUA AAA GU], [5′-H2PO4-(8-oxoG)(8-oxoG) CUA AAA GU], and [5′-H2PO4-(8-oxoG) CUA AAA GU]. The described experiments were carried out with 200 picomols of RNA (20 pmol used for MALDI analyses); however, lower amounts may result in detectable peaks with spectrometers using laser sources with more power than the one used in this work. Importantly, the described methodology can be generalized and potentially extended to product identification for other processes involving RNA and DNA, and may aid in the characterization/elucidation of other biochemical pathways.
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DOI：
10.1016/j.jbc.2021.101124
发表时间：
2021-10
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Ghodke PP;Guengerich FP
通讯作者：
Guengerich FP
影响因子：
5
作者：
Estevez M;Valesyan S;Jora M;Limbach PA;Addepalli B
通讯作者：
Addepalli B
影响因子：
5
作者：
Phillips CN;Schowe S;Langeberg CJ;Siddique N;Chapman EG;Resendiz MJE
通讯作者：
Resendiz MJE
影响因子：
15
作者：
Resendiz, Marino J. E.;Pottiboyina, Venkata;Sevilla, Michael D.;Greenberg, Marc M.
通讯作者：
Greenberg, Marc M.
DOI：
10.1002/cbic.202100521
发表时间：
2022-04-20
期刊：
Chembiochem : a European journal of chemical biology
影响因子：
--
作者：
通讯作者：
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