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，波罗斯R2的20微米微珠添加到每个样品为15分钟，在室温下搅拌。准备中止与1体积的甲醇1体积珠的“波罗斯珠，然后添加5％的甲酸/ 0.2％TFA的10卷。 </li><li>条件2 C 18 ZipTips吸入20μL0.1％TFA的尖端，把它推回去移液器。这样做两次，0.1％T的足总杯，四次，用70％乙腈/ 0.1％TFA的，和4倍，0.1％TFA的再次。 </li><li>条件C 18 ZipTips加载到C 18树脂在ZipTips的背后吸取珠浆和推用移液器体积的两个样本（控制和脱甲基反应）。 </li><li> 20μL0.1％TFA的两次洗ZipTips。 </li><li>洗脱在40μL70％乙腈/ 0.1％TFA的。 </li><li>完全用Speed​​Vac浓缩冻干每个洗脱。 </li></ol> 2。还原甲基化修改本协议的一部分，从布莱尔等。Rayment等。6,7。 <ol><li>重新暂停在100μL50 mM磷酸盐缓冲液，pH值7.4的脱甲基反应。 </li><li>要脱甲基反应，添加15毫克/毫升硼烷二甲胺8μL。硼烷二甲胺溶解在50 mM磷酸盐缓冲液，pH值7.4，给予15毫克/毫升原液。这应新鲜。 </li><li>到去甲基反应，添加16μL250毫米氘甲醛。 250毫米的氘甲醛准备稀释的股票，在H 2 0。这应新鲜。 </li><li>在4°C孵育2小时。 </li><li>重复步骤2.2，2.3和2.4。 </li><li>重复步骤2.2。 </li><li>孵育4°C的〜15小时。 </li><li>到去甲基样品，加12.5μL1M的Tris，pH值7.4解渴还原甲基化反应。 </li></ol> 3。电离质谱光谱法<ol><li>波罗斯R2的20微米微珠添加到脱甲基反应，并收集ZipTips步骤1.3-1.6所述。 </li></ol>注：重新挂起100μL50 mM磷酸盐缓冲液，pH值7.4的控制和治疗其他样品在3.1步开始。 <ol start="2"><li>洗脱到电离的样品板使用2μL33％饱和从ZipTips的控制和脱甲基反应样本ð2,5 - 二羟基苯甲酸。准备2,5 - 二羟基苯甲酸33％，使70％的乙腈/ 0.1％TFA的2,5 - 二羟基苯甲酸的饱和溶液，然后稀释至33％，70％乙腈/ 0.1％TFA的饱和。允许洗脱样品干燥和结晶。 </li><li>收集质谱使用的MALDI-prOTOF 1 PerkinElmerSciex质谱仪或提供高分辨率MALDI质谱8,9,10。 </li><li>查看光谱和提取使用PerkinElmerSciex TOFworks质谱仪制造商提供的软件或软件的峰面积。 </li><li>由MS验证可用质谱系统提供串联质谱功能的反应产物。 </li></ol> 4。数据分析<ol><li>为了弥补使用重型甲醛（图1B）创建的同位素重叠，确定峰面积（A）的13 C 2和碳同位素的相对论比e来控制样品，没有经过还原甲基化（图2A）的单一同位素峰。 公式1：一个一个12C / 13C2 = R 1 公式2：13C4 / 12C = R 2 这将使你在这些同位素的国家现有的肽比（R 1和R 2）具体的实验和质谱仪。 </li><li>使用下列公式R 1和R 2值量化的脱甲基反应样品的每个修改状态（图2B）在现有的肽的相对量： 公式3：H3K4me2 = 1 4式：H3K4me = 2 - R 1（1） 5式：H3K4的= 3 - R 2（1） - [R 1（2 - R 1（1））] </li></ol> 5。代表结果使用类LSD1，我们将展示为量化的MassSQUIRM的有效性赖氨酸去甲基化。如在协议文本部分中所述，我们进行了125纳克类LSD1和0.25微克二甲基化组蛋白H3肽（ARTKme2QTARKSTGGKAPRKQLYK-生物素）1去甲基化酶的检测。受到控制和脱甲基反应样品还原甲基化和MALDI质谱。控制反应，没有经过还原甲基表明，这种肽的典型的同位素信封和峰面积为方程1和2（图2A）。为脱甲基反应的质谱峰面积为方程3-5（图2B）提供。使用控制和脱甲基反应的峰面积，我们确定的肽类LSD1去甲基化给下面的反应产物：33.7％二甲基Lys4，单甲基Lys4 42.3％，24％未甲基化的赖氨酸4。参考布莱尔等。类LSD1去甲基化酶活性的全面分析以及抑制剂的研究6。注意变量的变化，如反应时间，酶浓度和底物浓度将允许活动的深入分析。 <img alt="图1" src="/files/ftp_upload/3604/3604fig1.jpg" /> 图1。MassSQUIRM的概述。 （一）从组蛋白H3 N端肽是被联合国（绿色），单（红色），或DI-赖氨酸4甲基化（蓝色）。在每个肽的化学成分的变化，导致差电离量化复杂。 （二）还原甲基化转换所有赖氨酸残基二甲基状态，这会导致所有肽同样电离。使用甲醛还原甲基化反应中的重，允许保留原甲基化的身份。开放各界表明轻甲基化而封闭的圆圈表示沉重的甲基化。 从布莱尔等人，2011年 6 修饰 。 <img alt="图2" src="/files/ftp_upload/3604/3604fig2.jpg" /> figurE 2。MassSQUIRM可以用来量化差异甲基化肽。 （一）一个DI-甲基化合成蛋白H3胜肽（ARTKme2QTARKSTGGKAPRKQLYK-生物素）分析用质谱和相对单一同位素峰的峰比值分别为R 1和R 2方程1和2中指出。 （二）同一合成肽孵育在37°C与125纳克在两个小时的脱甲基缓冲区类LSD1样品受到MassSQUIRM分析。一个叠嶂的混居的代表在图1B所示的三个不同的甲基化状态。单一同位素峰下面积分别记为1，A 2和A 3。峰面积被用来在方程3-5。开放各界表明轻甲基化而封闭的圆圈表示沉重的甲基化。 从布莱尔等修饰。 2011年 6。

<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>

我们什么都没有透露。

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 ...

application-masssquirm-for-quantitative-measurements-lysine

Research

JoVE Journal

Biology

13.3K 次观看.  University of Arkansas for Medical Sciences. 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。

视频: Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine 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 acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

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

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

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

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

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 ...

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

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

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

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

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

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

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

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

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

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

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

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 exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

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

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

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

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. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

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

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

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 ...

Particle Templated Emulsification enables Microfluidic-Free Droplet Assays

Reactions performed in monodispersed droplets afford enhanced accuracy and sensitivity compared to equivalent ones performed in bulk. However, the requirement of microfluidics to form controlled droplets imposes a barrier to non-experts, limiting their use. Here, we describe particle templated emulsification, an approach to generate monodisperse droplets without microfluidics. Using templating hydrogel spheres, we encapsulate samples in monodispersed droplets by simple vortexing. We demonstrate the approach by using it to perform microfluidic-free digital PCR.

Water-in-oil droplet assays are useful for analytical chemistry, enzyme evolution, and single cell analysis, but typically require microfluidics to form the droplets. Here, we describe particle templated emulsification, a microfluidic-free approach to perform droplet assays.