用肽微阵列分析组蛋白抗体特异性
Summary
该手稿描述了将肽微阵列技术应用于识别组蛋白及其翻译后修饰的抗体的特异性分析的方法。
Abstract
组蛋白蛋白的翻译后修饰（PTM）在调节染色质结构和基因表达方面的作用被广泛研究。组蛋白PTM特异性抗体的大规模生产和分布大大促进了对这些标记的研究。由于组蛋白PTM抗体是许多染色质生物化学应用的关键试剂，因此严格的抗体特异性分析对准确的数据解释和现场持续进展是必需的。该协议描述了用于分析组蛋白抗体特异性的肽微阵列的设计，制造和使用的集成管线。该方案的设计和分析方面由ArrayNinja（一种我们最近开发的开源和交互式软件包）来简化微阵列打印格式的定制。该管道已被用于筛选大量市售和广泛使用的组蛋白PTM抗体s，并且通过在线和扩展的组蛋白抗体特异性数据库免费获得从这些实验产生的数据。除组蛋白之外，本文描述的一般方法可广泛应用于PTM特异性抗体的分析。
Introduction
基因组DNA通过组蛋白蛋白质优雅地包装在真核细胞核内，形成染色质。染色质的重复亚基是核小体，它由围绕组蛋白的八聚体核心卷绕DNA的147个碱基对- H2A，H2B，H3和H4 1。染色质广泛地组织成松散包装的真空染色质和紧密堆积的异染色质结构域。染色质紧密程度调节了蛋白质机制能够访问底层DNA进行基本DNA模板过程（如复制，转录和修复）的程度。
在染色质的上下文中的基因组可访问的关键调节剂是对组蛋白2，3的非结构化尾和核心结构域翻译后修饰。组蛋白PTMs通过影响染色质4的结构并间接作用直接起作用h招聘读者蛋白质及其相关的大分子复合物，其具有染色质重塑，酶促和脚手架活动5 。组蛋白PTM功能在过去二十年的研究绝大多数都表明这些标记在调节细胞命运，生物体发育和疾病发生/进展中起关键作用。由于基于质谱的蛋白质组学技术的进步，已发现超过20种独特的组蛋白PTM超过80种不同的组蛋白残基6 。值得注意的是，这些修改往往发生在组合，并与“组蛋白密码”假设是一致的，大量的研究表明，读者蛋白质通过识别组蛋白PTM的7，8，9的特定组合的有针对性的染色质的离散区域。向前迈进的一个关键挑战将是将功能分配给gr组蛋白PTM的不完整列表，并确定组蛋白PTM的具体组合如何协调与染色质相关的动态功能。
抗体是用于检测组蛋白PTM的lynchpin试剂。因此，已经商业开发了用于染色质生物化学研究的超过1,000种组蛋白PTM特异性抗体。随着高通量DNA测序技术的快速发展，这些试剂被ChIP-seq（染色质免疫沉淀加上下一代测序）的个别研究者和大规模表观遗传学“路线图”计划（ 例如 ENCODE和BLUEPRINT）广泛使用）等的管道以产生组蛋白PTM分发的全基因组10，11的高分辨率空间地图。然而，最近的研究已经表明，组蛋白PTM抗体的特异性可能是高度可变的，并且这些试剂显示不合适 avorable性能如脱靶表位识别，由相邻的PTM强阳性和负面影响，以及难以在特定残基鉴别修饰顺序（ 例如 ，单- ，二- ，或三甲基赖氨酸）12，13，14，15 ，16，17，18。因此，组蛋白PTM特异性抗体试剂的严格质量控制对于准确解释用这些有价值的试剂产生的数据是必要的。
微阵列技术能够以高通量，可重现和小型化的格式同时询问成千上万的大分子相互作用。为此，已经创建了各种微阵列平台来分析蛋白质-DNA 19 ，“> 20，蛋白质21，和蛋白质-肽相互作用22。事实上，组蛋白肽微阵列已经成为一个信息发现平台染色质生物化学研究，使作家，橡皮擦的高通量分析，和翻译后修饰15组蛋白的读者，23，24，以及用于组蛋白抗体特异性的分析17，25。除了其在染色质和表观遗传学研究中的应用，组蛋白肽阵列具有潜在效用作为诊断/预后测试系统性红斑狼疮等自身免疫性疾病，其中抗染色质自身抗体产生26,27。
在这里，我们描述了我们为设计，制造和排队开发的集成管道使组蛋白肽微阵列产生识别组蛋白及其PTMs的抗体的特异性谱。我们最近开发的ArrayNinja是一个开源的交互式软件应用程序，其中集成了微阵列实验的设计和分析阶段28 。 ArrayNinja在Google Chrome中效果最好。简言之，机器人接触式微阵列打印机用于在链霉亲和素包被的玻璃显微镜载片上的限定位置沉积生物素 – 缀合的组蛋白文库。然后可以以竞争性和平行的测定形式使用数组来询问抗体 – 表位相互作用（ 图1 ）。肽文库由数百种独特的合成肽组成，分别含有PTM（赖氨酸乙酰化，赖氨酸/精氨酸甲基化和丝氨酸/苏氨酸磷酸化），以及大部分衍生自蛋白质组学数据集的相关组合。肽合成和验证方法在其他地方详细23 。使用该阵列平台从我们正在进行的组蛋白PTM抗体筛选工作产生的数据归档在公共网络资源 – 组蛋白抗体特异性数据库（www.histoneantibodies.com）上。值得注意的是，与此协议的变型制造的组蛋白肽的微阵列也已被广泛地用于表征组蛋白PTM读者域8，29，30，31，32，33，34，35，36，37的活性以及最近个人资料组蛋白PTM作家和橡皮擦活动24 。
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图1：组蛋白肽微阵列上抗体筛选的逐步程序的动画描述。具有确定的翻译后修饰（红色和蓝色圆圈）的生物素化组蛋白肽与生物素荧光素在链霉抗生物素蛋白包被的玻璃上共同印刷。正相互作用可视化为红色荧光。 请点击此处查看此图的较大版本。
Protocol
1.安装并运行ArrayNinja 从www.virtualbox.org下载并安装Oracle Virtual Box。 从http://research.vai.org/Tools/arrayninja下载并解压缩ArrayNinja虚拟机（VM）。 打开虚拟框，并通过单击“机器”，“添加”添加ArrayNinja VM，然后从保存了ArrayNinja VM的文件夹中选择arrayninja.vbox。 通过在Virtual Box中选择它并点击绿色的“开始”箭头启动ArrayNinja。 虚拟框将打开一个新窗口，并显示一条消息，…
Representative Results
该方案已被用于设计和制造用于分析组蛋白PTM抗体特异性的肽微阵列平台。该阵列查询了超过300个独特的肽特征（长度为20-40个残基）的文库，代表了核心和变体组蛋白38上发现的许多已知PTM组合。该管道已经成为筛选许多广泛使用和市售的组蛋白PTM抗体的主力，并且在组蛋白抗体特异性数据库（www.histoneantibodies.com）上提供了完整的数据集。 <sup class="xref…
Discussion
在生物医学研究中的应用抗体可靠性是最重要的46，47。在染色质生物化学中尤其如此，鉴于抗体的位置是开发用于表征组蛋白PTM的丰度和分布的大多数技术的关键工具。这里提出的方案详细介绍了用于分析组蛋白PTM抗体特异性的肽微阵列的设计，制造和使用的优化管线。该管道已被用于筛选大量市售和广泛使用的组蛋白PTM抗体，并且通过在线和扩展的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到Van Andel研究所的一部分支持，以及美国国家卫生研究院（CA181343）对SBR的研究资助
Materials
| Printing Buffer | ArrayIt | PPB | |
| BSA | Omnipure | 2390 | |
| Streptavidin-coated glass microscope slides | Greiner Bio-one | 439003-25 | |
| polypropylene 384 well plate | Greiner Bio-one | 784201 | |
| Biotin-fluorescein | Sigma | 53608 | |
| contact microarray printer | Aushon | 2470 | Aushon 2470 Microarray Printer |
| contact microarray printer | Gene Machines | OmniGrid 100 | OmniGrid Microarray Printer |
| PBS | Invitrogen | 14190 | |
| Blocking Buffer | ArrayIt | SBB | |
| Hydrophobic wax pen | Vector Labs | H-4000 | ImmEdge Hydrophobic Barrier PAP Pen |
| Silicon Gasket | Grace Bio-labs | 622511 | |
| Hybridization Vessel | Thermo Scientific | 267061 | or similar vessel |
| Fluorescent-dye conjugated secondary antibody | Life Technologies | A-21244 | Alexa Fluor 647 (anti-rabbit) |
| Fluorescent-dye conjugated secondary antibody | Life Technologies | A-21235 | Alexa Fluor 647 (anti-mouse) |
| Wax Imprinter | ArrayIt | MSI48 | |
| Tween-20 | Omnipure | 9490 | |
| Microarray Scanner | Innopsys | InnoScan 1100AL | or equivalent microarray scanner |
| EipTitan Histone Peptide Microarray | Epicypher | 112001 | |
| AbSurance Pro Histone Peptide Microarray | Millipore | 16668 | |
| MODified Histone Peptide Array | Active Motif | 13001 | |
| Histone Code Peptide Microarrays | JPT | His_MA_01 | |
| Wax | Royal Oak | GulfWax | for wax imprinter |
| Humidified Microarray Slide Hybridization Chamber | VWR | 97000-284 | |
| High throughput microscope slide washing chamber | ArrayIt | HTW | |
| Microscope slide centrifuge | VWR | 93000-204 | |
| Antibody 1 | Abcam | 8898 | |
| Antibody 2 | Millipore | 07-473 | |
| Biotinylated histone peptide | EpiCypher | 12-0001 | Example peptide. Similar peptides with various modifications are available from several commercial sources. |
| ImageMagick | https://www.imagemagick.org/script/index.php | ||
| ArrayNinja | https://rothbartlab.vai.org/tools/ |
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Cite This Article
Cornett, E. M., Dickson, B. M., Rothbart, S. B. Analysis of Histone Antibody Specificity with Peptide Microarrays. J. Vis. Exp. (126), e55912, doi:10.3791/55912 (2017).