Name: application of masssquirm for quantitative measurements of lysine demethylase activity
Uploaded: 2012-03-11T14:00:00
Description: Watch this Scientific Journal Video about Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity at JoVE.com

我们感谢质谱支持UAMS蛋白质组学设施。由NIH资助P20RR015569，P20RR016460和R01DA025755为这个项目提供了资金。

该协议被修改布莱尔等6。 1。类LSD1去甲基化检测<ol><li>在终体积20μL，结合0.25微克二甲基化组蛋白H3肽脱甲基缓冲区（ARTKme2QTARKSTGGKAPRKQLYK-生物素）（50毫米的Tris-Cl的pH值8.5，50 mM的氯化钾，氯化镁2 5毫米，5％的125纳克重组类LSD1甘油）。此外，执行没有酶肽单独控制。控制是第3和不需要还原的甲基化。 </li><li>在37°C孵育2小时</li><li>收集肽，8μL，波罗?...

<ol>
	<li>Goldberg, A. D., Allis, C. D., Bernstein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics:+a+landscape+takes+shape.">Epigenetics: a landscape takes shape.</a> Cell. 128, 635-638 (2007).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifications+and+their+function.">Chromatin modifications and their function.</a> Cell. 128, 693-705 (2007).</li><li>Blair, L. P., Cao, J., Zou, M. R., Sayegh, J., Yan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+Regulation+by+Lysine+Demethylase+5+(KDM5)+Enzymes+in+Cancer.">Epigenetic Regulation by Lysine Demethylase 5 (KDM5) Enzymes in Cancer.</a> Cancers (Basel). 3, 1383-1404 (2011).</li><li>Cole, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+probes+for+histone-modifying+enzymes.">Chemical probes for histone-modifying enzymes.</a> Nat. Chem. Biol. 4, 590-597 (2008).</li><li>Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+demethylation+mediated+by+the+nuclear+amine+oxidase+homolog+LSD1.">Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.</a> Cell. 119, 941-953 (2004).</li><li>Blair, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MassSQUIRM:+An+assay+for+quantitative+measurement+of+lysine+demethylase+activity.">MassSQUIRM: An assay for quantitative measurement of lysine demethylase activity.</a> Epigenetics. 6, 490-499 (2011).</li><li>Rayment, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+structure+of+myosin+subfragment-1:+a+molecular+motor.">Three-dimensional structure of myosin subfragment-1: a molecular motor.</a> Science. 261, 50-508 (1993).</li><li>Collom, S. L., Jamakhandi, A. P., Tackett, A. J., Radominska-Pandya, A., Miller, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CYP2E1+active+site+residues+in+substrate+recognition+sequence+5+identified+by+photoaffinity+labeling+and+homology+modeling.">CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling.</a> Arch. Biochem. Biophys. 459, 59-69 (2007).</li><li>Gradolatto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+cerevisiae+Yta7+Regulates+Histone+Gene+Expression.">Saccharomyces cerevisiae Yta7 Regulates Histone Gene Expression.</a> Genetics. 179, 291-304 (2008).</li><li>Taverna, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yng1+PHD+finger+binding+to+histone+H3+trimethylated+at+lysine+4+promotes+NuA3+HAT+activity+at+Lysine+14+of+H3+and+transcripiton+at+a+subset+of+targeted+ORFs.">Yng1 PHD finger binding to histone H3 trimethylated at lysine 4 promotes NuA3 HAT activity at Lysine 14 of H3 and transcripiton at a subset of targeted ORFs.</a> Mol. Cell. 24, 785-796 (2006).</li></ol>

MassSQUIRM是一种廉价单和二甲基赖氨酸去甲基化酶活性的综合分析和定量的方法。 MassSQUIRM提供定量不仅对产品的反应，但也为中间体。此法可以用来作为一个强大的工具学习类LSD1和其他组蛋白去甲基化酶的机制。这也将是有益的许多新发现PHF8如赖氨酸去甲基酶的分类，可用于某些甲基酶。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>类LSD1 </td><td> BPS的生物科技公司</td><td> 50100 </td></tr><tr><td> H3K4me2-肽素</td><td>准备中的房子</td><td>没有</td></tr><tr><td>波罗斯R2的20微米微珠</td><td>应用生物系统公司</td><td> 1-1129-06 </td></tr><tr><td> C 18 ZipTip </td><td> Millipore公司</td><td> ZTC18M </td></tr><tr><td>三氟乙酸（TFA） </td><td>热</td><td> 28904 </td></tr><tr><td>乙腈</td><td>费舍尔</td><td> A996 </td></tr><tr><td> 2,5 - 二羟基苯甲酸</td><td>西格玛</td><td> 85707 </td></tr><tr><td>硼烷二甲胺</td><td>西格玛</td><td> 180238 </td></tr><tr><td>同位素重D 2 -甲醛</td><td>剑桥同位素实验室</tD&gt; <td> DLM-805-20 </td></tr><tr><td>三</td><td>费舍尔</td><td> BP154 </td></tr><tr><td>氯化钾</td><td>费舍尔</td><td> BP366 </td></tr><tr><td> MgCl 2的 </td><td>费舍尔</td><td> BP214 </td></tr><tr><td>甘油</td><td>费舍尔</td><td> 33国集团</td></tr><tr><td>蚁酸</td><td> Fluka公司</td><td> 06440 </td></tr><tr><td>甲醇</td><td>费舍尔</td><td> A452 </td></tr><tr><td>钠 - 磷酸</td><td>费舍尔</td><td> BP329 </td></tr><tr><td> Speed​​Vac浓缩</td><td>学者</td><td> DNA110 </td></tr><tr><td> MALDI - prOTOF质谱仪和TOFworks的软​​件的</td><td> PerkinElmerSciex </td><td>没有</td></tr></tbody></table>

application of masssquirm for quantitative measurements of lysine demethylase activity

近日，后生监管机构已发现1-3在许多不同的疾病的关键球员。因此，这些酶的主要目标是为小分子的研究和药物开发4。许多后生监管机构最近才发现，仍然是在被分类的过程。在这些酶的赖氨酸去甲基从组蛋白和其他蛋白质的赖氨酸甲基组。由于这一类酶的小说性质，一些实验已经开发，研究他们的活动。这一直是一个路障，以分类和高吞吐量研究组蛋白去甲基化酶。目前，很少去甲基化酶检测存在。那些确实存在往往在本质上是定性的，不能同时不同赖氨酸甲基化状态之间的辨别（联合国，单，二和三）。质谱是常用来确定脱甲基酶活性，但目前质谱分析没有解决差异甲基化多肽是否不同的电离。差甲基化肽的电离，使甲基化状态比较困难，肯定不定量（图1A）。因此，可以检测不优化为脱甲基酶活性的全面分析。 在这里，我们描述的方法称为MassSQUIRM（质谱定量使用同位素还原甲基）是基于还原甲基氘甲醛强制所有赖氨酸是DI-甲基胺基，从而使它们基本上是相同的化学物质，因此，电离相同（图1B）。唯一的化学还原甲基化的差异，是氢和氘，这并不影响电离电离效率。 MassSQUIRM法是与联合国，单或二甲基赖氨酸去甲基反应产物具体。该法也适用于赖氨酸甲基转移给SA我的反应产物。在这里，我们使用相结合的还原甲基化化学和MALDI质谱测量类LSD1活动，赖氨酸去甲基化酶能够消除DI-和单甲基基团，合成肽基板5。此法是简单，容易服从任何与电离质谱在实验室或通过蛋白质组学设施的实验室。该法〜8倍的动态范围，很容易扩展到板格式5。

Watch this Scientific Journal Video about Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity at JoVE.com

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

我们提出了一个使用MALDI质谱和还原甲基化化学的方法来量化赖氨酸甲基化的变化。

赖氨酸去甲基化酶活性的定量测量中的应用的MassSQUIRM

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

Research

JoVE Journal

Biology

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

用于测量的活性测定大肠埃希氏菌诱导赖氨酸Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

科研

生物学

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

定量，实时分析，利用分子信标病​​变特定的细胞裂解液中的碱基切除修复活动

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Analyzing and Building Nucleic Acid Structures with 3DNA

核酸结构分析及建设3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

蛋白赖氨酸甲基转移使用SPOT肽阵列特异性分析

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

发展与表征<em&gt;体外</em&gt;微血管网络和内皮的定量测量的[Ca<sup&gt; 2+</sup&gt;]<sub&gt;我</sub&gt;和一氧化氮含量

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

电生理测量在中性转运体活性研究中的应用

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

用数据无关采集质谱法定量测定点特异蛋白赖氨酸乙酰化和琥珀酰化化学计量学

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...