Name: sample preparation for mass spectrometry-based identification of rna-binding regions
Uploaded: 2017-09-28T14:00:00
Description: Watch this Scientific Journal Video about Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions at JoVE.com

导流洞得到了塞尔学者计划,. 史密斯基金会 (C1404) 和3月的硬币基金会 (1-财政年度-15-344) 的支持。R01GM110174 和 nih R01AI118891 以及国防部授予 BC123187P1 的支持。刻录-T。得到了 NIH 培训补助金 T32GM008216 的支持。

1. mESCs 的文化和扩展 注意: 小鼠胚胎干细胞易于培养, 可以迅速扩展到生物化学实验所需的大量时间, 这得益于它们的快速循环。健康的 mESCs 每12小时两倍. 
<ol> <li> 将 mESCs 扩展到所需的糊板上的制介质 (如下所示) 在37和 #176 的组织培养箱中; C、5% CO 2 和 #62; 95% 湿度. 
	注: 3-5 的生物复制为 + 4 su 样品和4SU 控制应准备足够的板材。一个10厘米的板块 ( 即 5-...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA-binding+proteomes+from+yeast+to+man+harbour+conserved+enigmRBPs.">The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs.</a> Nat Commun. 6, 10127 (2015).</li><li>Conrad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+interactome+capture+of+the+human+cell+nucleus.">Serial interactome capture of the human cell nucleus.</a> Nat Commun. 7, 11212 (2016).</li><li>Wilusz, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+triple+helix+stabilizes+the+3'+ends+of+long+noncoding+RNAs+that+lack+poly(A)+tails.">A triple helix stabilizes the 3' ends of long noncoding RNAs that lack poly(A) tails.</a> Genes Dev. 26 (21), 2392-2407 (2012).</li><li>Kim, T. K., Shiekhattar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architectural+and+Functional+Commonalities+between+Enhancers+and+Promoters.">Architectural and Functional Commonalities between Enhancers and Promoters.</a> Cell. 162 (5), 948-959 (2015).</li><li>Brannan, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SONAR+Discovers+RNA-Binding+Proteins+from+Analysis+of+Large-Scale+Protein-Protein+Interactomes.">SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.</a> Mol Cell. 64 (2), 282-293 (2016).</li><li>Bonasio, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+with+RNA+direct+the+Polycomb+group+protein+SCML2+to+chromatin+where+it+represses+target+genes.">Interactions with RNA direct the Polycomb group protein SCML2 to chromatin where it represses target genes.</a> Elife. 3, e02637 (2014).</li><li>Kaneko, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+JARID2+and+noncoding+RNAs+regulate+PRC2+recruitment+to+chromatin.">Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin.</a> Mol Cell. 53 (2), 290-300 (2014).</li><li>Kaneko, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+the+PRC2+component+Ezh2+is+cell+cycle-regulated+and+up-regulates+its+binding+to+ncRNA.">Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA.</a> Genes Dev. 24 (23), 2615-2620 (2010).</li><li>Saldana-Meyer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTCF+regulates+the+human+p53+gene+through+direct+interaction+with+its+natural+antisense+transcript,+Wrap53.">CTCF regulates the human p53 gene through direct interaction with its natural antisense transcript, Wrap53.</a> Genes Dev. 28 (7), 723-734 (2014).</li><li>Castello, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Identification+of+RNA-Binding+Domains+in+Human+Cells.">Comprehensive Identification of RNA-Binding Domains in Human Cells.</a> Mol Cell. 63 (4), 696-710 (2016).</li><li>He, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Mapping+of+RNA-Binding+Regions+in+the+Nuclear+Proteome+of+Embryonic+Stem+Cells.">High-Resolution Mapping of RNA-Binding Regions in the Nuclear Proteome of Embryonic Stem Cells.</a> Mol Cell. 64 (2), 416-430 (2016).</li><li>Favre, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4-Thiouridine+photosensitized+RNA-protein+crosslinking+in+mammalian+cells.">4-Thiouridine photosensitized RNA-protein crosslinking in mammalian cells.</a> Biochem Biophys Res Commun. 141 (2), 847-854 (1986).</li><li>Hafner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome-wide+identification+of+RNA-binding+protein+and+microRNA+target+sites+by+PAR-CLIP.">Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.</a> Cell. 141 (1), 129-141 (2010).</li><li>Favre, A., Saintome, C., Fourrey, J. 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我们描述一个详细的实验协议, 以执行 RBR ID 在 mESCs, 并在适当的修改, 在任何细胞, 可以纳入4SU 到 RNA。其他的细胞类型可能需要优化的方法, 以确保足够的信噪比。此外, 虽然本文所述的议定书侧重于核限制性商业惯例的审查, 但 RBR ID 技术应易于适应不同的细胞隔间, 如胞或特定的器, 使用不同的分馏战略.可能需要优化的参数包括4SU 浓度和并网时间以及 UV 交联的能量。这些参数对于确保 RNA 蛋白?...

RBR ID 方法的目的是确定新的 rna 结合蛋白 (限制性商业惯例) 和映射他们的 rna 结合的区域 (RBRs) 与肽水平的决议促进 rna 结合的突变体的设计和研究生物和生物化学蛋白质-RNA 相互作用的作用。
RNA 在生物分子中是独一无二的, 因为它既能充当携带遗传信息的信使, 也可以折叠成复杂的三维结构, 其生物化学功能更类似于蛋白质的12。越来越多的证据表明, 编码 rna (rna) 发挥重要作用的各种基因调控和表观遗传途径3,4,5和, 通常, 这些管理功能是调解的演唱会与特定的 RNA 相互作用的蛋白质。特别重要的是, 一组相互作用的蛋白质最近被确定为强烈研究的长 ncRNA (lncRNA) Xist, 提供有价值的洞察如何这 lncRNA 介导的女性细胞 X 染色体失活6,7 ,8。值得注意的是, 这些 Xist 相互作用的蛋白质中有几个不包含任何规范的 rna 绑定域9, 因此它们的 rna 绑定活动不能根据它们的主序列单独被预测为在硅片中。考虑到数以千计的 lncRNAs 在任何给定的单元格10中表达, 有理由认为它们中的许多可能通过与尚未被发现的 RNA 结合蛋白 (限制性商业惯例) 的相互作用而行动。因此, 确定这些新的限制性商业惯例的实验战略将大大促进解剖 rna 生物功能的任务。
以往试图识别限制性商业惯例的经验依赖于波利亚 + RNA 选择耦合质谱 (MS)11,12,13,14,15。虽然这些实验在假定的限制性商业惯例中添加了许多蛋白质, 但通过设计他们只能检测到 polyadenylated 转录的蛋白质。然而, 大多数小 rna 和许多 lncRNAs 不是 polyadenylated 的。16,在这些实验中, 17及其相互作用的蛋白质可能会被遗漏。最近的一项研究应用机器学习蛋白质-蛋白质 interactome 数据库, 以识别蛋白质, co-purified 与多个已知的限制性商业惯例, 并表明这些经常性的惯例合作伙伴更有可能拥有 RNA 绑定活动18。然而, 这种方法依赖于挖掘现有的大型交互数据库, 并且只能识别可在变性条件下使用已知的限制性商业惯例 co-purified 的蛋白质, 从而排除不溶、膜嵌入和缺乏蛋白质.
将蛋白质鉴定为善意的限制性惯例通常不会自动产生关于蛋白质-RNA 相互作用的生物和/或生物化学功能的信息。为了解决这一点, 通常需要确定相互作用中所涉及的蛋白质域和氨基酸残基, 以便特定的突变体可以被设计成在每一种新的限制性条件下测试 RNA 结合的功能19,20. 我们的小组和其他人以前的努力使用重组蛋白片段和删除突变体来识别 RNA 结合区域 (RBRs)19,20,21,22;然而, 这种方法是 labor-intensive 和不相容的高通量分析。最近, 一项研究描述了使用质谱仪23以高通量方式绘制 RNA 结合活动的实验策略;然而, 这种方法依赖于双波利亚 + RNA 的选择, 因此与上面描述的限制性惯例识别方法具有相同的限制。
我们开发了一种技术, 称为 rna 结合区域识别 (RBR ID), 利用蛋白质 rna 光和定量质谱法来鉴定蛋白质和蛋白质区域与 RNA 在活细胞中的相互作用, 而不做假设在 RNA 多状态, 因而包括限制性商业惯例对波利亚-rna24。此外, 这种方法完全依赖于交联, 对蛋白质的溶解度或可达性没有要求, 因此适于在细胞膜内 (例如核包络) 或不太可溶性的隔间映射 RNA 结合活动 (例如核矩阵)。我们描述了对小鼠胚胎干细胞的细胞核进行 RBR 的实验步骤 (mESCs), 但稍加修改, 本协议应适用于多种细胞类型, 只要它们能有效地将4SU 从培养中.

<table>
	<tbody>
		<tr>
			<td>KnockOut DMEM</td>
			<td>Fisher Scientific</td>
			<td>10829018</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum, qualified, US origin</td>
			<td>Fisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine solution 200 mM</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential Amino Acid Solution (100&#215;)</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>ESGRO Leukemia Inhibitory Factor (LIF)</td>
			<td>EMD Millipore</td>
			<td>ESG1106</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris</td>
			<td>4423</td>
			<td></td>
		</tr>
		<tr>
			<td>PD0325901</td>
			<td>Sigma</td>
			<td>PZ0162</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin solution,2% in water</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td></td>
		</tr>
		<tr>
			<td>4-thiouridine</td>
			<td>Sigma</td>
			<td>T4509</td>
			<td>50 mM stock in water</td>
		</tr>
		<tr>
			<td>Spectrolinker XL-1500</td>
			<td>Fisher Scientific</td>
			<td>11-992-90</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride</td>
			<td>Sigma</td>
			<td>78830</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CO-630</td>
			<td>Sigma</td>
			<td>542334</td>
			<td>Commercial form of octyl-phenoxy-polyethoxy-ethanol detergent</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma</td>
			<td>I6125</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, sequencing grade</td>
			<td>Promega</td>
			<td>V5111</td>
			<td></td>
		</tr>
		<tr>
			<td>Empore solid phase extraction disk</td>
			<td>3M</td>
			<td>66883</td>
			<td></td>
		</tr>
		<tr>
			<td>OLIGO R3 Reversed - Phase Resin</td>
			<td>Fisher Scientific</td>
			<td>1133903</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>Sigma</td>
			<td>E8263</td>
			<td>High purity nuclease</td>
		</tr>
		<tr>
			<td>Sonic Dismembrator Model 100</td>
			<td>Fisher Scientific</td>
			<td>discontinued</td>
			<td>updated with FB505110</td>
		</tr>
		<tr>
			<td>HPLC grade acetonitrile</td>
			<td>Fisher Chemical</td>
			<td>A955-4</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC grade water</td>
			<td>Fisher Scientific</td>
			<td>W6 4</td>
			<td></td>
		</tr>
		<tr>
			<td>TFA</td>
			<td>Fisher Scientific</td>
			<td>A11650</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Bicarbonate</td>
			<td>Sigma</td>
			<td>A6141</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma</td>
			<td>49199</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Sigma</td>
			<td>F0507</td>
			<td></td>
		</tr>
		<tr>
			<td>ReproSil-Pur 18-AQ</td>
			<td>Dr. Maisch GmbH HPLC</td>
			<td>r13.aq.0003</td>
			<td>Packing material for HPLC column</td>
		</tr>
		<tr>
			<td>Capillary for nano columns (75 &#181;m)</td>
			<td>Molex</td>
			<td>1068150017</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQuant software</td>
			<td>Max Planck Institute for Biochemistry</td>
			<td></td>
			<td>Can perform chromatographic alignment of multiple MS runs</td>
		</tr>
	</tbody>
</table>

sample preparation for mass spectrometry-based identification of rna-binding regions

编码 rna 在几个核过程中起重要作用, 包括调节基因表达、染色质结构和 DNA 修复。在大多数情况下, 编码 rna 的作用是由蛋白质介导的, 其功能反过来受这些相互作用与编码 rna。与此相一致的是, 越来越多的涉及核功能的蛋白质被报告为结合 rna, 在少数情况下, 这些蛋白质的 rna 结合区域已经被映射, 通常是通过费力的 candidate-based 方法。
在这里, 我们报告一个详细的协议, 以执行高通量, 蛋白质组范围内的 rna 结合蛋白和它们的 rna 结合区域的无偏见的识别。该方法依赖于在细胞 rna 中加入 photoreactive 苷模拟, 其次是紫外介导的蛋白质-rna 交联, 以及质谱分析, 以揭示蛋白质组内的 rna 交联肽。虽然我们描述了小鼠胚胎干细胞的过程, 但该协议应该很容易适应于各种培养细胞。

图 1描述了 RBR ID 工作流。由于这种技术的交联效率相对较低, 因此在生物复制中考虑到观察效果 (P-值) 的损耗水平和一致性非常重要。图 2显示了 RBR ID 结果的火山图。重叠 RNA 识别主题 (RRM) 域的多肽显示出高度一致的损耗水平。RRM 域可用作 RBR 分析的正则控制。
RBR ID 可用于...

Watch this Scientific Journal Video about Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions at JoVE.com

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

我们描述了一个协议, 以确定 rna 结合蛋白和地图他们的 rna 结合区域的活细胞使用紫外介导的光和质谱。

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 ...

Research

JoVE Journal

Biochemistry

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

A Tandem Liquid ChromatographyÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œMass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

对于代谢物分析基于法 - 串联液相色谱 - 质谱法<em&gt;金黄色葡萄球菌</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

科研

生物化学

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 ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

用奇米蛋白构建功能蛋白区的鉴定

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

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 ...

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

使用自动工作站进行质谱测量的等离子体样品制备

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

探头电喷电镀质谱的样品制备

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

无目标液相色谱-质量光谱学-小麦粒基代谢代谢学分析

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

蛋白质组学设施提交和后续数据分析的TMT样品制备

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view ...

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

VMXm 光束线微晶样品制备管道

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

基于质谱的蛋白质组学方法用于全局和高置信度的蛋白质R-甲基化分析

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...