Name: identification of rna fragments resulting from enzymatic degradation using maldi-tof mass spectrometry
Uploaded: 2022-04-11T14:00:00
Description: Watch this Scientific Journal Video about Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry at JoVE.com

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。

本研究采用不含RNase的超纯水（表1）。
1. RNA溶液的浓度测定
<ol><li>按照以下步骤制备RNA样品。<ol>
<li>使用微量离心管（0.6 mL）通过将1μL储备溶液（ 通过 固相合成获得）19稀释到159μL 无RNase H2O中来制备RNA溶液。通过将混合物反复上下移液（10x）来混合溶液。 注意：由于市面上有各种各样的比色皿，因此所需的体?...

<ol>
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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.6 mL MCT Graduated Violet</td>
			<td>Fisher Scientific</td>
			<td>05-408-127</td>
			<td></td>
		</tr>
		<tr>
			<td>6&#8217;-Trihydroxyacetophenone monohydrate 98%</td>
			<td>Sigma Aldrich</td>
			<td>480-66-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile 99.9%, HPLC grade</td>
			<td>Fisher Scientific</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine triphosphate, 10 mM</td>
			<td>New Englang Bioscience</td>
			<td>P0756S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium citrate, dibasic 98%</td>
			<td>Sigma Aldrich</td>
			<td>3012-65-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Fluoride 98.0%, ACS grade</td>
			<td>Alfa Aesar</td>
			<td>12125-01-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Bruker bacterial test standard</td>
			<td>Bruker Daltonics</td>
			<td>8255343</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial source of Xrn-1</td>
			<td>New England BioLabs</td>
			<td>M0338S</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl pyrocarbonate, 97%</td>
			<td>ACROS Organics</td>
			<td>A0368487</td>
			<td></td>
		</tr>
		<tr>
			<td>Flex analysis software</td>
			<td>Bruker daltonics</td>
			<td></td>
			<td>FlexAnalysis software version 3.4, Bruker Daltonics</td>
		</tr>
		<tr>
			<td>Lambda 365 UV-vis spectrophotometer</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI plate: MSP 96 ground steel target</td>
			<td>Bruker Daltonics</td>
			<td>280799</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass Spectrometer</td>
			<td>Bruker</td>
			<td></td>
			<td>Microflex LRFTOF mass spectrometer (Bruker Daltonics, Billerica, MA)</td>
		</tr>
		<tr>
			<td>Mili-Q IQ 7000</td>
			<td>Milipore Sigma</td>
			<td></td>
			<td>A Mili-Q system was used to purify all water used in this work</td>
		</tr>
		<tr>
			<td>Nanosep Centrifugal Devices with OmegaTM Membrane 10 K, blue (24/pkg)</td>
			<td>Pall Corporation</td>
			<td>OD010C33</td>
			<td>filter media, Omega (modified polyethersulfone) 10 K pore size</td>
		</tr>
		<tr>
			<td>NEBuffer 3</td>
			<td>New England Biolabs</td>
			<td>B7003S</td>
			<td>This is solution B</td>
		</tr>
		<tr>
			<td>Oligo Analyzer tool</td>
			<td>IDT-DNA</td>
			<td></td>
			<td>https://www.idtdna.com/calc/analyzer</td>
		</tr>
		<tr>
			<td>Pipette tips P10</td>
			<td>Fisher Scientific</td>
			<td>02-707-441</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips P200</td>
			<td>Fisher Scientific</td>
			<td>02-707-419</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Away</td>
			<td>Molecular BioProducts</td>
			<td>7005-11</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 Polynucleotide Kinase</td>
			<td>New England BioLabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 Polynucleotide Kinase Reaction Buffer</td>
			<td>New England BioLabs</td>
			<td>B0201S</td>
			<td>This is solution A</td>
		</tr>
		<tr>
			<td>Triflouroacetic Acid</td>
			<td>Alfa Aesar</td>
			<td>76-05-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Xrn-1 exoribonuclease</td>
			<td>Expressed in house</td>
			<td>See ref. 20</td>
			<td></td>
		</tr>
		<tr>
			<td>ZipTip Pipette Tips for Sample preparation</td>
			<td>Millipore</td>
			<td>ZTC 18S 096</td>
			<td>10 &#181;L pipette tips loaded with a C18 standard 0.6 &#181;L bed</td>
		</tr>
	</tbody>
</table>

identification of rna fragments resulting from enzymatic degradation using maldi-tof mass spectrometry

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。

本工作中使用的寡核苷酸在使用前被合成，表征和定量。所有寡核苷酸的浓度 通过 在90°C下记录的紫外可见光谱测定，以避免因二级结构的潜在形成而产生的错误读数。 图3 显示了本工作中使用的RNA的模型寡核苷酸的光谱，在室温下和加热后拍摄。
整个过程以及导致Xrn-1失速的氧化病变的结构如图 4所示。寡核苷酸（1）在?...

Watch this Scientific Journal Video about Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry at JoVE.com

Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

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。

使用MALDI-TOF质谱法鉴定酶降解产生的RNA片段

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, ...

质谱法已被广泛用于各种应用。本文中的方法提供了从体外酶促过程获得的RNA片段的明确表征。该技术的优点包括获得分子铁峰，可以使用少量材料进行实验以进行体外应用，并且可以通过最少的训练来完成方案。所描述的方法可以广泛应用于研究涉及DNA，RNA或蛋白质的过程。示例包括生化转化，例如交叉信息，化学修饰的鉴定或其他生物聚合物相互作用和反应性。一个困难可能发生在获得MALDI光谱仪或提供此服务的核心设施上。另一个方面是确定检测限，根据仪器的不同，检测限可能更高或更低。首先，打开紫外可见分光光度计，然后单击珀金埃尔默UV WinLab图标以打开仪器操作控制窗口。使用仪器右侧的开关打开帕尔贴温度控制器分光光度计，然后单击“基本方法”下的 Scan-Lambda 365。将打开另一个屏幕，提示用户确保电池座为空。单击“确定”并允许仪器进行系统检查。现在，通过选择“数据收集”来调整扫描参数。在新窗口中的“扫描设置”下，将起点更改为 350 纳米，将终点更改为 215 纳米。通过选择配件左侧的加号来激活珀尔帖。然后单击“多细胞帕尔贴”，将温度摄氏度更改为25摄氏度，然后单击“帕尔贴开”，导航到样品信息选项卡，输入所需的样品数量、名称和细胞位置。单击“自动归零”并插入包含背景解决方案的比色皿。单击“开始”（Start） 并将比色皿插入所需的单元格中，并确保比色皿窗口的方向平行于仪器的正面。现在，单击“确定”以25摄氏度开始第一次扫描。要在较高温度下进行测量，请单击多孔帕尔贴，将温度更改为所需的样品温度并重复扫描。单击“文件”，“导出”并选择所需的文件位置，然后选择“XY 数据”以获取文本文件。为了进行RNA的5'磷酸化，通过加入33.5微升无RNA酶的水，5微升溶液A，6微量10毫摩尔三磷酸腺苷，1微量200微摩尔RNA水溶液和4.5微升多核苷酸激酶在0.6微升微量离心管中制备溶液，并轻轻混合溶液。将反应混合物置于水浴中，在37摄氏度下孵育45分钟。然后通过将反应管置于加热块中来灭活酶，在65摄氏度下预热10分钟。让溶液冷却至室温，以产生最终的5'磷酸化RNA溶液。孵育后，使用50微升该溶液，加入5微升溶液B和5微升Xrn-1的1-飞托莫溶液进行RNA水解。将反应管在室温下孵育两小时。将该反应混合物转移到10千道尔顿孔径离心装置中，并通过以700倍G离心10分钟过滤酶。然后，加入20微升无RNase的水以洗涤离心管上的残留RNA，然后离心。最后，将该滤液与先前通过在10千道尔顿孔径离心装置中离心得到的所得滤液结合，并将溶液在零摄氏度或船舶上冷冻以进行分析。要对样品进行脱盐和浓缩，请使用市售的阳离子交换C18移液器吸头，其末端装有C18色谱介质床。为此，将10微升移液器吸头放在10微升移液器上，并用50%乙酰丁腈水溶液清洗该吸头两次。每次将用过的体积丢弃到单独的废物管中，并用10微升0.1%三氟乙酸水溶液平衡C18尖端两次。从移液器中手动取出C18吸头，并将其固定在装有P200移液器吸头的200微升移液器上。然后将C18尖端浸入Xrn-1水解RNA寡核苷酸期间制备的所得溶液中，并吸出溶液通过C18尖端释放10次。现在，从P200移液器中取出C18吸头，将其定位到10微升移液器中，并使用0.1%三氟乙酸溶液的水溶液洗涤C18吸头两次。每次将用过的体积丢弃到单独的废液管中。用10微升无RNase的水清洗C18尖端两次。为了在MALDI板上发现，通过将样品浸入所需的基质中并将溶液分配回管中10次，从C18尖端洗脱RNA寡核苷酸。将此溶液移出到板上两个单独的点上，每个点一微升，并允许光斑风干。这项工作中使用的RNA浓度是通过在90摄氏度下记录的紫外可见光谱测定的，以避免由于二级结构的潜在形成而产生的错误读数。事件的总体顺序包括两个部分。一，RNA的高效5'磷酸化，仅出现一个对应于预期产物的峰来证明。二、失速片段由用Xrn-1处理样品混合物而产生的。使用包含两个氧化病变的不同序列获得相同的结果。需要考虑的潜在变化包括消除滤波装置的使用或考虑使用不同的矩阵进行MALDI点测。例如，西那酸或吡啶甲酸。RNA片段是从酶促过程中获得的。该工艺是通过耦合MALDI，电泳分析，固相合成和圆二向色性建立的。所描述的协议有可能用于各个领域，从化学科学研究到生物医学领域的应用。

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Unit 1

Analyzing Cells and Proteins

SCIENTISTS IN ACTION

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

科研

生物化学

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

脂滴隔离定量质谱分析

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

MALDI 质谱法测定小鼠组织提取物蛋白酶特异性: 操作 PH 值导致特异性改变

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

用质谱和电镜分析 Biolayer 干涉传感器上的动态蛋白复合物的组装和释放

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

使用开源 MALDI TOF-MS IDBac 管道分析微生物蛋白和专用代谢物数据

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

血管紧张蛋白II图解的硝酸分析与鉴定

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...