使用MALDI-TOF质谱法鉴定酶降解产生的RNA片段
Summary
MALDI-TOF用于表征从氧化RNA和外切核糖核酸酶Xrn-1之间的反应性中获得的片段。本方案描述了一种可应用于涉及RNA和/或DNA的其他过程的方法。
Abstract
RNA是存在于生命所有领域的生物聚合物，它与其他分子和/或反应物质（例如DNA，蛋白质，离子，药物和自由基）的相互作用无处不在。结果，RNA经历各种反应，包括其分裂，降解或修饰，导致具有不同功能和含义的生物学相关物种。一个例子是鸟嘌呤氧化成7，8-二氢-8-氧代嘌呤（8-氧代嘌呤），这可能在活性氧（ROS）存在下发生。总体而言，表征此类产品和转化的程序对科学界来说非常有价值。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。本方案描述了如何表征酶处理后形成的RNA片段。所选择的模型使用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核苷酸长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]，通过紫外可见光谱定量， 并通过 MALDI-TOF进行表征。然后将获得的链（1）5′-磷酸化 并通过 MALDI-TOF表征;（2）用Xrn-1处理;（3）过滤脱盐;（4） 通过 MALDI-TOF进行分析。该实验装置明确鉴定了与Xrn-1失速相关的片段：[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]。所描述的实验是用200皮摩尔RNA（20 pmol用于MALDI分析）进行的;然而，较低的量可能会导致光谱仪使用比本工作中使用的功率更大的激光源来检测峰。重要的是，所描述的方法可以推广，并可能扩展到涉及RNA和DNA的其他过程的产物鉴定，并可能有助于表征/阐明其他生化途径。
Introduction
MALDI-TOF1，2，3 是一种广泛使用的技术，用于表征和/或检测不同大小和特征的分子。它的一些用途包括各种应用，例如检测自然资源4中的单宁，对食品中的代谢物进行成像5，发现或监测细胞药物靶标或标志物6，以及临床诊断7，仅举几例。与本工作相关的是将MALDI-TOF与DNA或RNA一起使用，其在寡核苷酸上的使用可以追溯到三十多年前8，其中注意到了几个局限性。该技术现已发展成为一种可靠的常用手段，用于表征生物聚合物9 并鉴定/理解化学和生化反应，例如，表征RNA10中的铂化位点，鉴定链裂解11，12后的RNA片段或蛋白质 – DNA交联的形成13.因此，说明和突出显示使用此技术的重要方面是有价值的。MALDI-TOF的基础知识也以视频格式进行了描述， 本文将不再赘述。此外，其在DNA或蛋白质上下文中的应用先前已以所述格式15，16，17描述和说明。
本文报道了用于检测酶水解后形成的RNA片段的方案。该实验模型是根据我们第18组最近发表的一项发现选择的，其中MALDI-TOF用于确定含有氧化病变8-oxoG的RNA的外切核糖核酸Xrn-1和寡核苷酸之间的独特反应性。 通过 固相合成19，[5′-CAU GAA ACA A（8-oxoG）G CUA AAA GU]和[5′-CAU GAA ACA A（8-oxoG）（8-oxoG）CUA AAA GU]获得20-核苷酸长链，而Xrn-1则按照先前描述的报告20表达和纯化。简而言之，Xrn-121 是一种5′-3’外切核糖核酸酶，具有各种关键的生物学作用，可降解多种类型的RNA，包括氧化RNA22。结果表明， 8-oxoG时酶的合成能力失速， 导致RNA片段含有5′-磷酸化末端[5′-H2PO4-（8-oxoG）G CUA AAA GU]，[5′-H2PO4-（8-oxoG）CUA AAA GU]和[5′-H2PO4-（8-oxoG）CUA AAA GU]18.
最后，重要的是要注意质谱是一种强大的方法，通过各种方法，可以适应其他目的23，24;因此，选择正确的电离方法以及其他实验设置至关重要。
Protocol
Representative Results
Discussion
该工作流程中的主要挑战出现在完成实验和进行质谱分析之间。实验在科罗拉多大学丹佛分校进行并完成，并（一夜之间）运往科罗拉多州立大学的设施。根据方便，在收到数据后进行数据采集。一些意外情况导致该过程的时间延迟。在一种情况下，意外的仪器故障要求样品在发现和采集之前被冷冻（一次21天）;然而，这种时间延迟似乎并不影响实验结果，尽管不能排除RNA的降解（导致信号强度?…
Declarações
The authors have nothing to disclose.
Acknowledgements
值得注意的是，这项工作是三个机构，两个研究小组和一个核心设施之间的合作努力。分布和工作量如下进行：蛋白质（Xrn-1）表达在丹佛大学（科罗拉多州丹佛市）进行。寡核苷酸的合成，定量和实验（主要是酶降解）在科罗拉多大学丹佛分校（科罗拉多州丹佛市）进行。那里也进行了优化。MALDI-TOF发现，采集和分析在科罗拉多州立大学的分析资源核心设施中进行。（科罗拉多州柯林斯堡）。SS希望感谢UROP奖（CU Denver）和Eureca赠款（CU Denver）的支持。E. G.C. 通过 R00GM115757 感谢 NIGMS 的支持。MJER 通过 1R15GM132816感谢NIGMS的支持。K.B. 确认资源 ID： SCR_021758。这项工作还得到了亨利·德雷福斯基金会（Henry Dreyfus Foundation）的教师学者奖（MJER）TH-21-028的支持。
Materials
| 0.6 mL MCT Graduated Violet | Fisher Scientific | 05-408-127 | |
| 6’-Trihydroxyacetophenone monohydrate 98% | Sigma Aldrich | 480-66-0 | |
| Acetonitrile 99.9%, HPLC grade | Fisher Scientific | 75-05-8 | |
| Adenosine triphosphate, 10 mM | New Englang Bioscience | P0756S | |
| Ammonium citrate, dibasic 98% | Sigma Aldrich | 3012-65-5 | |
| Ammonium Fluoride 98.0%, ACS grade | Alfa Aesar | 12125-01-8 | |
| Bruker bacterial test standard | Bruker Daltonics | 8255343 | |
| Commercial source of Xrn-1 | New England BioLabs | M0338S | |
| Diethyl pyrocarbonate, 97% | ACROS Organics | A0368487 | |
| Flex analysis software | Bruker daltonics | FlexAnalysis software version 3.4, Bruker Daltonics | |
| Lambda 365 UV-vis spectrophotometer | Perkin Elmer | ||
| MALDI plate: MSP 96 ground steel target | Bruker Daltonics | 280799 | |
| Mass Spectrometer | Bruker | Microflex LRFTOF mass spectrometer (Bruker Daltonics, Billerica, MA) | |
| Mili-Q IQ 7000 | Milipore Sigma | A Mili-Q system was used to purify all water used in this work | |
| Nanosep Centrifugal Devices with OmegaTM Membrane 10 K, blue (24/pkg) | Pall Corporation | OD010C33 | filter media, Omega (modified polyethersulfone) 10 K pore size |
| NEBuffer 3 | New England Biolabs | B7003S | This is solution B |
| Oligo Analyzer tool | IDT-DNA | https://www.idtdna.com/calc/analyzer | |
| Pipette tips P10 | Fisher Scientific | 02-707-441 | |
| Pipette tips P200 | Fisher Scientific | 02-707-419 | |
| RNase Away | Molecular BioProducts | 7005-11 | |
| T4 Polynucleotide Kinase | New England BioLabs | M0201S | |
| T4 Polynucleotide Kinase Reaction Buffer | New England BioLabs | B0201S | This is solution A |
| Triflouroacetic Acid | Alfa Aesar | 76-05-1 | |
| Xrn-1 exoribonuclease | Expressed in house | See ref. 20 | |
| ZipTip Pipette Tips for Sample preparation | Millipore | ZTC 18S 096 | 10 µL pipette tips loaded with a C18 standard 0.6 µL bed |
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