房颤是由欧洲研究理事会的欧盟第七框架计划下，开始资助项目（FP7 / 2007-2013）/ ERC资助协议N°203413和密涅瓦中心生物混合复杂systems.HA和AI支持由达莉亚和丹公司Maydan奖学金为高级学位的学生在耶路撒冷的希伯来大学。

1.设计一个多肽阵列<ol><li>划分目标蛋白的序列插入部分重叠的10-20残基的肽。变化重叠在特定实验的数量和执行实验室的资源，但原则上重叠的时间越长越好。当设计的肽考虑到在蛋白质帐户已知的二级结构元件，可以是负责的相互作用。 </li><li>以便通过商业供应商所设计肽阵列。这里，使用的肽共价结合到通过C末端的阵列。 </li></ol> 2.阻断非特异性吸附<ol><li>使含有0.05％Tween 20（TBST / PBST）中的50mM的Tris或磷酸盐缓冲溶液，并调节到所需的pH用HCl或NaOH的（为了知道精确的离子强度）的测量的量和所需要的离子强度用NaCl 。这里，可以使用pH为7.5和150mM的离子强度。 </li><li>使封闭液2.5％（重量/体积）的TBST / PBST中的脱脂奶粉。 </li><li>为了防止非特异性结合，将其浸入阵列在5ml封闭溶液。孵育所述阵列为2-4小时，在室温下或过夜，在4ºC振荡器上。 </li></ol> 3.与蛋白质孵化<ol><li>用5毫升封闭液30秒，然后两次用5毫升TBST / PBST中5分钟，在摇动器上在室温下的第一洗阵列。 </li><li>孵育洗涤阵列用5ml含2.5％（重量/体积）脱脂奶粉，以防止非特异性结合的His标记的蛋白质溶液。这里，使用4.5的蛋白溶液微米（STIL 500-650）溶解在所述的封闭溶液。 注意:一般5-10微米蛋白质被用于筛查，但蛋白质浓度可以甚至低至2-3微米，这取决于结合亲和力和局部浓度下结合的阵列上的肽（效率的合成）。缓冲器，其中蛋白质溶解可以改变，但通常使用如上所述的相同的封闭溶液稀释的蛋白质。 </li><li>孵育的蛋白溶液的阵列为3-8小时，在室温下或过夜，在4ºC。 </li></ol> 4.与抗体孵育<ol><li>用5毫升TBST / PBST中洗涤阵列三次。第一次洗涤是30秒，随后用两个5分钟的洗涤在摇床上于室温。 </li><li>孵育洗涤阵列用5ml稀释的HRP偶联的抗体（1:1500），在包含相同的成分在相同浓度的封闭溶液，1小时在摇床上于室温温育缓冲液中。该抗体可结合任一靶蛋白或标签。 </li><li>通过重复相同的协议，而不STE进行对照实验在阵列上测试该抗体的相互作用的肽，特别是如果该数组包含从靶蛋白（ 例如 ，用于低聚的研究）的肽第3页，与蛋白质孵育。 </li><li>用5毫升TBST / PBST中洗涤阵列三次。第一次洗涤是30秒，随后用两个5分钟的洗涤在摇床上于室温。 </li></ol> 5.读阵列<ol><li>开展化学发光法的发展与ECL免疫印迹底物试剂盒。 </li><li>用发光图像分析仪进行检测。 </li><li>分析结果。确保该阵列上的两个重复显示相同的信号，因此，结果是可靠的。如果蛋白伴侣的结构是已知的，搜索该结构的结合肽，并查找结合位点。甚至肽远的顺序可以并拢的三级结构，并创建一个结合位点。 </li></ol>

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The authors have nothing to disclose.

多肽阵列筛选是一个极好的工具，用于识别其合作伙伴靶蛋白的结合位点。该测定法是快速和容易地进行，其结果可以在一个或两个工作日内得到。肽阵列筛选是通用的，并且可以用于多种用途。它可以被用于表征交翻译修饰如磷酸化，并确定激酶的底物。例如:FLT3激酶，它与急性骨髓性白血病，磷酸化通过肽阵列筛选6发现含酪氨酸的肽底物。酪氨酸激酶抑制剂帕唑帕尼和拉帕替尼的抑制性质，使用的肽阵列筛选23进行测试。肽阵列可用于鉴定介导蛋白质 - 蛋白质相互作用的位点。这样的蛋白质的研究中使用这种方法我们实验室的例子是:所述的HIV-1的Vif蛋白和细胞A3G蛋白之间的相互作用进行了表征我们荷兰国际集团肽阵列24;肽阵列筛选用于研究抗凋亡BCL-2家族的相互作用与促凋亡ASPP2蛋白25，并研究tBID，BCL-2家族的成员的结合，与线粒体蛋白MTCH 2 26;利用肽阵列筛选27球蛋白的IIC结构域之间的分子内相互作用进行了分析。在这里，我们提出了我们如何使用肽阵列筛选鉴定介导的STIL-CHFR相互作用，蛋白质-蛋白质相互作用的增殖和癌症的14重要场所的一个例子。揭示的精确结合位点可以作为依据所研究的相互作用的抑制剂的合理设计。 肽阵列筛选不是一个定量分析。它是半定量的，因为肽在所述阵列上的每个点的数量而变化，由于在合成收率和肽的纯度差。这反过来又依赖于pept中IDE序列，长度等的产量和纯度的变化也有时会导致假阴性或假阳性结果。同一阵列内的强度之间的比较可以进行，导致半定量等级。不同阵列或不同实验之间。然而，这样的比较用相同的阵列是不可靠的。量化的结合，在所述肽序列的筛选中发现的肽应该被合成并测试了使用生物物理方法，如荧光各向异性，等温滴定量热法（ITC），表面等离子体共振（SPR） 等。这样的测定法是在目标蛋白的结合不总是敏感到弱的结合，因为它们需要高浓度的蛋白质，以检测这种结合。肽阵列筛选，在另一方面，是要弱结合非常敏感。因此，一些在阵列筛选中观察到的相互作用不能被使用的方法，例如荧光anisot观察或定量<html>粘稠14。该肽阵列筛选需要非常低浓​​度的蛋白结合测定法的灵敏度高的事实使得所述肽序列用于筛选在宽范围的亲和力蛋白 - 肽相互作用的有用方法。 假阴性结果也可能是由于这一事实，即线性肽可能无法正确​​地表示在该蛋白质的结合位点的三维结构。然而，这是不与内在无序蛋白，其通过肽很好表示的问题。假阳性结果，也可以通过抗体结合到阵列引起的。这些可以通过如在协议4.3节中所述进行的对照实验来检测。假阴性结果，也可以得到，如果由该抗体识别的蛋白的表位成为结合后的肽阵列的未曝光。此问题是不常见的，但可以通过使用多克隆抗体对蛋白质加以解决。 jove_content"&gt;肽阵列筛选，也可以用于提高引线的肽，包括丙氨酸扫描和SAR研究7。一旦目标的结合位点是已知的，根据它的序列的阵列可被设计成具有多种变型，包括更换各残余物丙氨酸（丙氨酸扫描）以确定重要的残基的相互作用。以类似的方式，合成孔径雷达的研究可以通过改变结合肽的不同性质来进行，其中包括不同的肽的长度，由相应的D取代的残基α-氨基酸和其他修饰，例如甲基化，糖基化和取代的非天然氨基酸。这种修饰的肽，都基于一个靶序列，可以在一个阵列上进行合成和筛选的靶蛋白结合在一个快速和容易的实验，提供了丰富的信息，用于提高潜在抑制剂和理解了相互作用的分子机制。 <p class="jove_content"&gt;肽阵列筛选的方法是高度灵活的和变化中的协议可以在需要时被应用。乳旁其他封闭化合物，如牛血清白蛋白（BSA）或蔗糖，都可以使用。如果非特异性结合时观察到的，应该使用更高浓度的牛奶/ BSA /蔗糖。该蛋白可与任何期望的缓冲阵列一起温育，但要添加到缓冲器牛奶/ BSA /蔗糖或用于在阻断溶液中的任何其他阻断剂，以防止非特异性结合是很重要的。蛋白质浓度应保持相对低，并且强的结合甚至可以用3-5微米的蛋白进行观察。培养中的所有步骤（粘连，温育与蛋白质或抗体，洗涤）的体积应足以完全覆盖阵列。通常4-5毫升就足够了。如果第一抗体是检测不到的次要抗体都可以使用。在这种情况下，步骤6和7，应重复进行稀释和培养时间适合特定的二级抗体。在检测过程依赖于抗体。辣根过氧化物酶（HRP）标记的抗体，化学发光是常用的在这个协议中说明。不同的方案可用于不同的抗体，例如荧光或电-化学发光法4。本文中描述的肽阵列仅用于单次使用，并且不能被重新使用。 这里介绍的协议表明肽阵列的广泛实用性。利用正确设计的阵列中，这两种蛋白之间的结合位点，可以进行详细的鉴定。一旦该结合位点被很好地表征它可以用来作为一种药物靶标位点，抑制相关的蛋白 - 蛋白相互作用。基于在所述阵列筛选中发现的结合肽，抑制性小分子可被设计和筛选药物动态。

蛋白 - 蛋白相互作用介导的大部分过程中的活细胞。受损的蛋白质相互作用可导致疾病，使得蛋白相互作用重要的药物靶标。因此，这是理解这些相互作​​用在分子水平上非常重要。蛋白质相互作用所使用的各种技术，从细胞和生物化学测定以定量的生物物理测定法的研究，并且这些可被执行或者与全长蛋白，用蛋白质结构域或肽。肽作为优秀的工具来研究蛋白质相互作用。这是因为肽可以容易地合成，并允许集中于特定的相互作用位点，一方面与多个蛋白质靶在另一方面1,2-以高通量方式进行。肽阵列筛选是一种快速，容易获得在短时间内3大量关于靶蛋白与许多伴侣的相互作用数据的执行方法。不像其他的生化或生物物理方法，用于检测和分析蛋白质 - 蛋白质相互作用，肽阵列筛选，需要非常低的蛋白质的浓度，并且可以检测到非常微弱的结合。肽阵列可用于多种应用中的肽-蛋白质相互作用，如蛋白-蛋白或受体-配体相互作用位点4，同源或异源寡聚化接口的映射，表征抗体的表位5，学习酶活6和高通量构筑物效关系（SAR）研究7。为深入审查有关肽阵列筛选看到卡茨等人 4 几种类型的肽阵列的当前存在的。有用于制备多肽阵列两大合成策略:合成肽它们附着于固相载体上的肽的，或合成直接在固体支持物上，主要是使用SPOT技术4,8-之前。该肽是由9-芴基（Fmoc）化学8在固体载体上合成通常。其中常见的合成方案是通过N端的肽连接（ 例如，JPT pepstar阵列9）和肽附着通过C末端（ 如 PEPSCAN pepchip阵列10，JPT pepspot阵列9和INTAVIS celluspot阵列11）4等。固体支持物可以有所不同，所以没有肽偶联到它的化学性质。半胱氨酸终止的肽可以通过硫醇基团12被连接到载玻片上。的N末端 ​​的肽可以通过氨基酸的酯化共价结合到纤维素上的膜具有羟基附8。 这里，我们提出了详细的方案用于筛选肽阵列作为用于研究蛋白质 - 蛋白质相互作用的方法。我们所使用的阵列是Celluspots阵列，它是一种含有大摩在微阵列UNT通过载玻片支撑在一个小的纤维素膜的斑点（高达384点的重复）的。这使蛋白质的低量和抗体的工作，并取得显著量为每一次实验的数据。该阵列还包含高肽密度可以检测低亲和力结合。该阵列被用于映射STIL-CHFR相互作用，这是用于控制正常细胞的增殖13非常重要。这两种蛋白之间的不受控制的相互作用可以导致癌症的发展。通过映射这种互动，我们发现了特异结合位点，并结合残基14。这种铺平了道路设计能抑制这种蛋白质 - 蛋白质相互作用理性抑制剂。

<table><tbody><tr><td>Peptide array</td><td>INTAVIS Bioanalytical Instruments</td><td></td><td></td></tr><tr><td>His-probe Antibody (H-3) HRP</td><td>Santa Cruz Biotechnology</td><td>sc-8036 HRP</td><td></td></tr><tr><td>EZ-ECL Kit</td><td>Biological industries</td><td>20-500-120</td><td></td></tr><tr><td>Skim Milk</td><td>Becton, Dickinson and Company</td><td>232100</td><td></td></tr><tr><td>LAS-3000 camera&amp;nbsp;</td><td>FUJI film</td><td></td><td></td></tr></tbody></table>

identifying protein-protein interaction sites using peptide arrays

蛋白 - 蛋白相互作用介导的大部分过程中的生物体的活细胞和控制的动态平衡。受损的蛋白质相互作用可导致疾病，使得蛋白相互作用重要的药物靶标。因此，这是理解这些相互作​​用在分子水平上非常重要。蛋白质相互作用所使用的各种技术，从细胞和生物化学测定以定量的生物物理测定法的研究，并且这些可被执行或者与全长蛋白，用蛋白质结构域或肽。肽作为优秀的工具来研究蛋白质相互作用，因为肽可以容易地合成，并允许针对特定的相互作用位点。多肽阵列使两种蛋白之间的相互作用位点的识别，以及筛选的靶蛋白结合用于治疗目的的肽。它们还允许高通量SAR的研究。对于识别的结合位点，一个typi的卡尔肽阵列通常包含部分地重叠，从所希望的靶蛋白的一种或多种伴侣蛋白的完整序列的10-20个残基的肽。筛选阵列对靶蛋白的结合揭示了结合肽，对应于在伴侣蛋白仅使用少量的蛋白质的结合位点，以容易和快速的方法。 在这篇文章中，我们描述了一个协议，用于筛选肽阵列映射的目标蛋白及其合作伙伴之间的互动网站。肽阵列是基于伴侣蛋白考虑到它们的二级结构的序列设计。在这个协议中使用的阵列制备了INTAVIS生物分析仪器Celluspots阵列。该阵列被阻断，以防止非特异性结合，然后与所研究的蛋白温育。使用抗体检测显示对应于该蛋白之间的特异性相互作用位点的结合的肽。

STIL是一个非常重要的中心体蛋白。它控制着细胞的正常分裂和细胞增殖13,15 - 19。 STIL有几种蛋白质18,20,21交互，而大部分的相互作用的发生，通过它的中心部分，这是一种本质上无序区（IDR）14。我们设计了一个由STIL结合蛋白CHFR衍生肽组成的数组。 CHFR]是诱导响应于有丝分裂应力22肿瘤抑制。因为CHFR的STIL结合结构域的结构是未知的时候，我们将所述蛋白质域到15个残基长的肽具有的7个残基的重叠，如在协议步骤1.1中描述。我们进行的肽阵列筛选用于识别CHFR-STIL结合位点，详见上述方案和图1所示，我们使用一个INTAVIS Celluspot阵列中重复组成384肽，它可以提供一个<html>巨大的信息量就证明在图2中，利用里找到的STIL的结合位点在它的结合蛋白CHFR（ 图3）该肽阵列。每个黑点在数组中表示CHFR衍生肽束缚STIL。请注意，每个肽中出现两次，因为它存在于重复的，这证实了结果的可靠性。 STIL和CHFR之间的相互作用在蛋白质水平13,14先前证实。肽阵列筛选揭示7 CHFR衍生肽，它们界定STIL的IDR，残基500-650（ 图3，Amartely 等人14）。这些结果使我们能够映射STIL对CHFR的结合位点。尽管这些7肽对CHFR一级序列的距离他们创建CHFR一个良好定义的结合位点的STIL（ 图4）14。 <html> 7 / 52097fig1highres.jpg"/&gt; 图1:肽阵列筛选的方案 A-设计，它包括来自一个或多个伙伴蛋白的部分地重叠肽的肽序列，考虑到它们的二级结构;。 B-与靶蛋白孵育; C-与抗体检测孕育; D-检测（可以通过化学发光，荧光等来完成）; E-分析结果揭示了蛋白质的结合位点。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52097/52097fig1large.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52097/52097fig2highres.jpg" /> 图2:多肽阵列筛选检测的示例用化学发光法取得的幻灯片中包含两个相同的数组为重复。ES / ftp_upload / 52097 / 52097fig2large.jpg"TARGET ="_空白"&gt;请点击这里查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/52097/52097fig3highres.jpg" /> 图3:筛选肽阵列STIL IDR结合CHFR蛋白:4.5微米His标签的STIL 500-650筛选结合数组。每个黑点表示STIL片段和CHFR衍生肽14之间的结合。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52097/52097fig3large.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52097/52097fig4highres.jpg" /> 图4:CHFR的STIL结合位点上的CHFR富含半胱氨酸结构域的结构如图（残基424-661，PDB ID:2XPO）七。肽结合的STIL在肽阵列筛选可以划分到粉红色，橙色和红色三个区域，它们是遥远的一级序列，但是并拢在三维结构中产生一个结合位点。这个全地区形成了STIL的二聚体CHFR 14结合口袋。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52097/52097fig4large.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Identifying Protein-protein Interaction Sites Using Peptide Arrays at JoVE.com

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

identifying-protein-protein-interaction-sites-using-peptide-arrays

Universidad San Sebastian

Research

JoVE Journal

Biology

17.9K 次观看.  The Hebrew University of Jerusalem. Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

视频: Identifying Protein-protein Interaction Sites Using Peptide Arrays

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

用肽微阵列分析组蛋白抗体特异性

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

科研

生物化学

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

肽为基础的识别功能图案和具有约束力的合作伙伴

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

免疫与感染

<meta charset="utf8"></head><body>预测蛋白质-蛋白质结合界面的氨基酸偏好

Computational Prediction of Amino Acid Preferences of Potentially Multispecific Peptide-Binding Domains Involved in Protein-Protein Interactions

Many protein-protein interactions involve the binding of short protein segments to peptide-binding domains. Usually, such interactions require the recognition of linear motifs with variable conservation. The combination of highly conserved and more variable regions in the same ligands often contributes to the multispecificity of binding, a common property of enzymes and cell signaling proteins. Characterization of amino acid preferences of peptide-binding domains is important for the design of mediators of protein-protein interactions (PPIs). Computational methods are an efficient alternative to the often costly and cumbersome experimental techniques, enabling the design of potential mediators that can be later validated in downstream experiments. Here, we described a methodology using the Pepspec application of the Rosetta molecular modeling package to predict the amino acid preferences of peptide-binding domains. This methodology is useful when the structure of the receptor protein and the nature of the peptide ligand are both known or can be inferred. The methodology starts with a well-characterized anchor from the ligand, which is extended by randomly adding amino acid residues. The binding affinity of peptides generated this way is then evaluated by flexible-backbone peptide docking in order to select the peptides with the best predicted binding scores. These peptides are then used to calculate amino acid preferences and to optionally compute a position-weight matrix (PWM) that can be used in further studies. To illustrate the application of this methodology, we used the interaction between subunits of human interferon regulatory factor 5 (IRF5), previously known to be multispecific but globally guided by a short conserved motif called pLxIS. The estimated amino acid preferences were consistent with previous knowledge about the IRF5 binding surface. Positions occupied by phosphorylatable serine residues exhibited a high frequency of aspartate and glutamate, likely because their negatively charged side chains are similar to phosphoserine.

<meta charset="utf8"></head><body>作者聚焦:一种破译多特异性蛋白质-蛋白质相互作用中氨基酸偏好的计算方法

We describe a methodology based on sequence diversification to estimate the amino acid preferences of multispecific binding sites in protein-protein interactions (PPIs). In this strategy, thousands of potential peptide ligands are generated and screened in silico, thus overcoming some limitations of available experimental methods.

参与蛋白质-蛋白质相互作用的潜在多特异性肽结合结构域的氨基酸偏好的计算预测

Many protein-protein interactions involve the binding of short protein segments to peptide-binding domains. Usually, such interactions require the ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

的蛋白质相互作用的合作伙伴在哺乳动物细胞中使用SILAC-免疫定量蛋白质组学鉴定

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

生物学

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

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 ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

选择反应监测质谱法进行绝对定量蛋白质

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

鉴定蛋白质相互作用的网站使用多肽阵列

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用肽微阵列分析组蛋白抗体特异性

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

肽为基础的识别功能图案和具有约束力的合作伙伴

参与蛋白质-蛋白质相互作用的潜在多特异性肽结合结构域的氨基酸偏好的计算预测

参与蛋白质-蛋白质相互作用的潜在多特异性肽结合结构域的氨基酸偏好的计算预测

参与蛋白质-蛋白质相互作用的潜在多特异性肽结合结构域的氨基酸偏好的计算预测

的蛋白质相互作用的合作伙伴在哺乳动物细胞中使用SILAC-免疫定量蛋白质组学鉴定

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

蛋白赖氨酸甲基转移使用SPOT肽阵列特异性分析

选择反应监测质谱法进行绝对定量蛋白质