This work has been supported by the DFG grant JE 252/7.

1.准备肽阵列<ol><li>该肽序列编程<ol><li>设计用于使用MultiPep去污剂软件的合成肽序列。输入单字母代码的肽序列。 肽1：TARKSTGGKA </li><li>生成丙氨酸或精氨酸步行库与特定命令（.replace，A）中筛选所需的识别一个定义衬底的重要氨基酸。例如在下面的例子中，第一行是野生型序列和来自第二行的每个氨基酸的肽被改变为丙氨酸顺序。 代替，A TARKSTG 一个 -ARKSTG T- 一个 -RKSTG TA-à-KSTG 焦油一个 -STG TARK-à-TG TARKS- 一个 -G TARKST- 一个 </li><li>使用类似的命令（.replace R，。代替n 等 ）在1.1.2与所有的天然可得的氨基酸残基（最终省略半胱氨酸，蛋氨酸和色氨酸所描述的，因为这些残基是容易氧化，有时降低合成产率）以合成肽阵列，用于确定共识底物序列基序的酶。 注意： 如图4A中，水平轴表示在给定的肽序列，纵轴表示被选择为命令的氨基酸。类似于在1.1.2中所示的序列中，A为纵轴意味着一个丙氨酸依次替换图1中的第一行中的给定的序列中的每个位置。 4A。同样精氨酸依次替换等与其它氨基酸的每个氨基酸在第二行中。完整的肽阵列库通过与各次的系统替换每个残基提供的肽底物序列的合成E的其他自然可用氨基酸残基，这有助于了解每个残基的在给定序列中的每个位置上的影响力。 </li><li>可替换地，已知的或预测的肽底物的甲基化可以容易地验证与PKMT的靶肽一起与阳性和阴性对照的甲基化合成肽文库（ 即公知的甲基化或已知不被甲基化的肽）。通过交换推定靶赖氨酸为丙氨酸，以确认目标赖氨酸的甲基化，如下所示通过手工编程合成肽。 野生型肽STGGķPRQFL 突变肽：STGG 一个 PRQFL 注：该软件还具有固有的脚本来创建不同的库，例如表位映射的序列所需的帧转移到地图需要交互的重要氨基酸。 </li></ol></li><li>膜的制备，氨基酸的ð试剂<ol><li>预膨胀的DMF（N，N-二甲基甲酰胺）10分钟的SPOT膜，然后将合成的SPOT框架上的湿膜没有气泡或皱纹。 </li><li>洗膜三次，100％的乙醇。在开始合成程序之前完全干燥该膜为至少10分钟。 </li><li>并联，新鲜制备的0.5M工作市售的Fmoc保护的氨基酸的浓度通过在NMP中溶解它们，也如表1中所述制备的活化剂和碱。 </li><li>确保SPOT合成器的所有废溶液的容器排空和再填充的溶剂容器与DMF，乙醇和哌啶。 注：现在的机器准备的合成。 </li></ol></li><li>第一个氨基酸的斑点<ol><li>以生成酰胺键与在膜上的游离氨基，激活呼入氨基酸的羧基基团由广告丁的活化剂（N，N&#39;-Diisopropylcarbodiimide）和碱溶液（乙基肟氰基）的氨基酸衍生物。 </li><li>当场对使用可编程机器人的氨基的PEG官能化膜活化的氨基酸的所需的卷。 注：由此，氨基酸的C末端被连接到所述隔膜的氨基。 </li><li>重复此步骤3次，以确保更好的第一活化的氨基酸偶联。孵育该膜20分钟，然后用DMF洗涤以除去未偶联的氨基酸。 </li></ol></li><li>非现场封锁区免费空间的 注：所有的肽的第一C端氨基酸，以该膜的氨基的偶联后，斑点和一些光点的区域内的氨基之间的氨基不形成与氨基酸债券。 <ol><li>通过与加帽溶液孵育阻断这些游离氨基（在DMF中的20％乙酸酐）5分钟。 注意：这避免了在进一步的周期与膜代替的生长的肽链的氨基酸的耦合。 </li></ol></li><li>除去Fmoc基团的<ol><li>封闭后，用DMF中的4倍洗膜，然后用20％哌啶孵育在DMF中20分钟以除去Fmoc保护基。 注意：此步骤导致除去氨基保护的Fmoc基团，它被称为去保护。 </li><li>之后，用乙醇两次用DMF和两次洗膜并使其干燥。 注：现在该膜是准备的下一个氨基酸的偶联。 </li></ol></li><li>链延伸 注：加入每个氨基酸被称为一个周期。除了第一个氨基酸偶联，所述周期开始用Fmoc基的偶联氨基酸的脱保护和它后面是传入氨基酸的活化的羧基的不断增长的PEP的氨基偶联潮链。 <ol><li>重复步骤1.3和1.5，直至所需肽的长度来实现的。 </li></ol></li><li>染色 注：在最后一次循环后，将肽阵列沾有溴酚蓝，以确认成功地合成肽。溴酚蓝结合与肽的游离氨基。肽斑点显示浅蓝色染色后，但根据不同的肽序列的点的强度而变化。这种染色可以确认完全除去哌啶的，因为在哌啶溴酚蓝的存在不染色的肽。此外，这也有助于标记为进一步使用肽阵列。 <ol><li>在最后的合成循环的Fmoc基团脱保护后，用DMF和100％的乙醇洗膜。然后，治疗与溴酚蓝（在DMF中的0.02％溴酚蓝），该膜为至少5分钟，直到它完全变为蓝色。 </li><li>之后，用DMF和ETH洗膜anol随后干燥10分钟。现在拿出从合成膜，进行侧链去保护（1.8节）手动。如果需要的话，拍摄该膜以记录肽合成（ 图2）的结果。 </li></ol></li><li>侧链脱保护<ol><li>处理该膜具有20至25ml侧链脱保护混合物由95％三氟乙酸（TFA）以裂解侧链保护基团和清除剂的试剂（2.5％水和2.5％三异丙基硅烷）保护的氨基酸侧链从在该步骤中修改。取脱保护混合物的足够体积，以确保它覆盖膜完全在密闭的化学抗性盒和孵化它为1至2小时，轻轻振动。 </li><li>用20-25毫升DCM（二氯甲烷）中的每个2分钟洗膜6次，最后两次用100％乙醇和干燥它在干燥器过夜。 注：现在该膜可用于实验。肽阵列合成的方案在图1中被提供。 </li></ol></li></ol> 2.蛋白质表达与纯化<ol><li> PKMTs为GST的融合蛋白表达<ol><li>使用标准技术，克隆编码全长PKMT或其催化域到细菌表达载体（如质粒pGEX-6P2），以将它表示为GST-融合蛋白的基因。 </li><li>传输与所需的PKMT载体插入BL21或BL21密码加E.大肠杆菌细胞通过热休克方法或任何其它方法。 </li><li>制备预培养用30ml的Luria-BERTANI（LB）培养基上表达的天孵育，在37℃下进行7至8小时，以连续摇动。 </li><li>接着，转移的10ml预培养到含有1升LB培养基的2L大挡板烧瓶中并孵育37 °C的孵化器连续摇晃，直到达到文化ES在600nm确定的光密度。 注意：虽然这可以为每个蛋白进行优化，我们经常诱导在约0.8的OD 600nm处 。诱导温度对每个单独的蛋白质进行优化，一些蛋白质显示出良好的表达在22 ℃，一些在较高的温度。 </li><li>转移细胞到感应温度15分钟，然后用诱导异丙基-β-D-硫代半乳糖苷为1mM，并允许培养中生长10-12小时，在诱导温度。 </li><li>之后通过离心收获细胞，在5000×g离心15分钟，最后用30ml的STE缓冲液（10mM的Tris pH值8.0，100mM的氯化钠和0.1mM EDTA）并储存在-20℃用于进一步使用洗涤沉淀。 </li></ol></li><li>蛋白质纯化<ol><li>解冻的细胞沉淀，并重新悬浮在30ml超声缓冲液（50mM的Tris pH值7.5，150mM的氯化钠，1mM的DTT和5％甘油）中，并通过超声处理破坏。 注：此缓冲区n电火工品必须为每个蛋白最终优化。 </li><li>离心细胞裂解物在22000×g离心1小时，并且，以后收集上清液并通过含有600微升的谷胱甘肽琼脂糖4B树脂的柱中。 </li><li>用50ml的超声缓冲液和下一个用100毫升高盐缓冲液（50mM的Tris pH值7.5，500mM的氯化钠，1mM的DTT和5％甘油）以除去非特异性结合的蛋白质的洗柱。洗脱结合的蛋白质用5毫升含40毫摩尔的谷胱甘肽高盐缓冲液中。 </li><li>透析洗脱的蛋白在2L具有低甘油（20毫摩尔Tris pH值7.4，100mM的氯化钾，0.5mM的DTT和10％甘油）中2小时，之后在20mM Tris pH值7.4，100mM的氯化钾，0.5mM的透析缓冲液DTT和70％甘油8小时或过夜。确保所有的纯化缓冲液是在4℃下，并在冷室中进行蛋白质纯化，以避免蛋白质变性。 </li></ol></li></ol> 3.肽阵列甲基化<ol><li>预INCU肽阵列插管<ol><li>执行肽阵列甲基无论是在一个盒子适当大小或在一个塑料袋，以避免酶和昂贵的放射性标记的AdoMet的浪费。预孵育的肽阵列膜在密封的塑料袋中含有不含酶的相应的甲基化缓冲液和标记的[甲基 - 3 H] -AdoMet 10分钟。缓冲器的体积取决于该阵列的大小，例如使用8个毫升甲基缓冲区满特异性轮廓的肽阵列。优化的量与所述的AdoMet的浓度每种酶。 </li></ol></li><li>肽阵列甲基化<ol><li>丢弃预孵育缓冲液孵育包含各自PKMT膜在8ml甲基缓冲液和标记的[甲基 - 3 H] -AdoMet为1至2小时。 注：需要的甲基化反应的酶量取决于具体PKMT的活动中，我们通常开始我们的筛选实验用50纳米酶片人集中。 </li></ol></li><li>肽阵列洗<ol><li>随后丢弃在放射性废物容器中的甲基化缓冲液，用20ml含有100mM碳酸氢铵和1％SDS缓冲液洗肽阵列为5次，每次5分钟，以除去结合的蛋白质。丢弃在放射性废物容器的洗涤液。 </li></ol></li><li>检测的放射性信号<ol><li>在洗涤后孵育该膜5分钟在10毫升扩增溶液（NAMP100V）。 </li><li>然后，丢弃扩增溶液进入放射性废物容器有机溶剂，密封在一个塑料袋肽阵列并把它放在一个纸盒放射自显影。 </li><li>将放射自显影胶片在黑暗的房间肽阵列上。仔细关闭磁带和暴露-80 °下所需的时间。然后，开发对结果进行分析的膜。捕捉图像几次不同博览会的n次，以避免强甲基化的肽底物饱和。 </li></ol></li></ol> 4.数据处理和分析<ol><li>现货强度的光密度分析<ol><li>扫描该膜在传统的扫描仪和通过光密度分析的斑点强度。做到这一点使用ImageJ（这是免费）或商业计划。 注：我们使用Phoretix软件这一任务。 </li></ol></li><li>规范化和结果从不同的膜的平均<ol><li>进行肽阵列甲基实验至少重复三次。正常化扫描强度为每个实验通过背景减除和设置的活性在参考衬底作为1.0。平均化的数据为各个斑点并报告它们作为平均值和标准偏差。 </li></ol></li><li>数据分析和显示<ol><li>绘制使用灰度或彩色方案来表示的相对活性在二维的数据。做到这一点的Wi日Excel或SIGMAPLOT如下图所示。此外，准备反复实验的标准偏差的分布曲线来分析数据的质量。 </li><li>比较和定量显示了认可，在底物肽的每个残基的精度，计算PKMT的每个氨基酸我在位置x由判别因子D 16,17的相对优先级： 二维X; I = &lt;VĴ≠I&gt; / V I - 1 其中v i是所述肽的变体携带I氨基酸在位置x和甲基化的速率&lt;VĴ≠I&gt;是所有19肽携带不同氨基酸Ĵ≠我在位置x的甲基的平均速率（包括野生型序列）。 注：本定义的歧视因素取决于本底水平。如果只有一个残基被接受在某一位置与背景的3％，则判定得到32.3因素。没有偏好的位置处造成的0的识别因素。 </li></ol></li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scansite+2.0:+Proteome-wide+prediction+of+cell+signaling+interactions+using+short+sequence+motifs.">Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs.</a> Nucleic Acids Research. 31, 3635-3641 (2003).</li><li>Takahashi, M., Ueno, A., Mihara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+design+based+on+an+antibody+complementarity-determining+region+(CDR):+construction+of+porphyrin-binding+peptides+and+their+affinity+maturation+by+a+combinatorial+method.">Peptide design based on an antibody complementarity-determining region (CDR): construction of porphyrin-binding peptides and their affinity maturation by a combinatorial method.</a> Chemistry. 6, 3196-3203 (2000).</li><li>Kramer, 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=Spot+synthesis:+observations+and+optimizations.">Spot synthesis: observations and optimizations.</a> J. Pept. Res. 54, 319-327 (1999).</li><li>Stadler, V., 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=Combinatorial+synthesis+of+peptide+arrays+with+a+laser+printer.">Combinatorial synthesis of peptide arrays with a laser printer.</a> Angew Chem. Int. Ed. Engl. 47, 7132-7135 (2008).</li><li>Schnack, C., Hengerer, B., Gillardon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+substrates+for+Cdk5+and+new+targets+for+Cdk5+inhibitors+using+high-density+protein+microarrays.">Identification of novel substrates for Cdk5 and new targets for Cdk5 inhibitors using high-density protein microarrays.</a> Proteomics. 8, 1980-1986 (2008).</li><li>Mah, A. 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=Substrate+specificity+analysis+of+protein+kinase+complex+Dbf2-Mob1+by+peptide+library+and+proteome+array+screening.">Substrate specificity analysis of protein kinase complex Dbf2-Mob1 by peptide library and proteome array screening.</a> BMC Biochemistry. 6, 22 (2005).</li></ol>

No conflicts of interest declared.

如这里所描述的SPOT合成是一个功能强大的方法来映射蛋白质 - 蛋白质相互作用位点，并调查肽修饰酶的底物识别。然而，SPOT合成仍有某些缺点，因为虽然由点法合成的肽被报道具有90％以上的纯度21这难以确认在每个实例。因此，结果必须通过其他方法，例如使用蛋白质结构域或与通过标准方法合成的纯化的肽被再现。另外，点阵列的另一主要缺点是，大多数时候它们不能被重新使用来?...

在过去的二十年里，若干报告证实在细胞发育和一些疾病，如癌症，但最近的蛋白赖氨酸的甲基化已成为一个重要的另一个PTM翻译后修饰（PTM）的重要性。而最初组蛋白赖氨酸的甲基化被认为是一个重要的染色质标记，以后的工作中也表现出若干非组蛋白1-4的赖氨酸的甲基化。甲基的从S-腺苷-L-甲硫氨酸的顺序转印到赖氨酸残基的ε氨基用一个家族的酶称为蛋白赖氨酸甲基转移（PKMTs）的包含超过60蛋白在人类基因组催化。 PKMTs最初发现的组蛋白修饰酶的具体甲基化赖氨酸残基，但后来的报告显示，他们也可以非甲基化，组蛋白5。到现在为止，大约5000赖氨酸甲基化位点被确定在不同的蛋白质6 </s向上&gt;，但负责这些修饰的酶经常没有确定。其中一个原因是，大多数PKMTs的特异性尚未得到广泛研究。因此，它循环地发生时PKMTs的新颖底物被发现。的PKMTs的底物特异性的详细知识的缺乏阻碍了它们的生物学功能和作用的理解。详细研究一个PKMT的特异性，即不同的一个或几个氨基酸多肽底物的甲基化率，必须测量和比较，这是理想的完成利用肽阵列。基于所得到的特异性概况，PKMTs的潜在底物可以被识别，可以进一步进行研究。 肽阵列是广泛使用的工具为抗体，肽修饰酶和蛋白质-蛋白质相互作用位点（抗体-抗原，受体-配体）7-9映射的生化分析。都需要SUC几百肽ħ应用。不同的方法可用于肽合成，其中肽在树脂上合成是很常用的，但它有局限性的吞吐量，这是相对昂贵的。这些问题得到解决，推出了弗兰克和他的同事10的SPOT合成方法。现货合成方法合成允许几百肽并行，平均也比树脂合成低廉。合成在纤维素膜上的肽可以使用，也可以直接用于各种应用或肽可以从膜被切割并在溶液测定中使用作为游离肽或以制备肽微阵列10-13。 SPOT合成的固相肽合成，其使用纤维素膜作为固体支持物，并采用标准的Fmoc-化学10-13的变体。因此，肽链的合成开始于C-末端，并进入朝所述N-末端在对比的生物合成的核糖体。纤维素膜官能化的第一个被激活的氨基酸（ 图1）的附件。当场方法是基于使用自动吸移系统的连续输送的活化氨基酸中的溶剂，以在所述膜限定的斑点的液滴。液体的液滴分配在那里形成一个圆形的湿斑，后来作为一个开放的反应器用于在肽合成的化学反应的多孔膜。光斑尺寸由分配的体积，膜的吸收能力确定的，这样的点的倍数被布置为阵列。合成的规模相关的光斑尺寸和隔膜的负载能力。斑点和阵列的密度之间的距离是通过改变该斑点尺寸管理。纤维素膜具有几个优点如在肽合成的固相，它是便宜的，耐受性的CHEMIC在肽合成中使用ALS，稳定在水溶液中，并容易处理。另外，它的亲水性使其适用于多种生物测定系统。 SPOT合成可以手动进行或自动（对于肽1000），这取决于肽的所需数量。从Intavis（科隆，德国）一个完全自动化的SPOT合成器可用于我们的应用程序。它允许合成肽不同长度的不同数量和。线性肽经常与15至20个氨基酸长度的合成，在高达42个氨基酸加成肽也可以通过逐步合成14,15制备。然而，增加的氨基酸的数目导致降低整体耦合的产量，从而影响所述肽的质量。因为每点的肽的低量的，该产品往往难以纯化和个别的肽的质量不能容易地进行评估。因此，结果从SPOT PEPT获得IDE阵列必须确认或者用肽通过标准方法在较大的规模，这可以被纯化，并根据在肽合成或通过合成含有所需的肽序列的蛋白质的标准分析合成。尽管如此，我们发现SPOT合成是高度可靠和一般结果是重复的。 SPOT合成不限于蛋白氨基酸，几种市售修饰的氨基酸也可用于合成，使肽之前和侧链保护基团的最终切割，此外后进行修改，它也允许掺入磷酸化，甲基化或乙酰化氨基酸11。 由斑点方法合成固定化肽文库可直接用于许多生物和生化分析。我们采用包含300-400肽进行调查PKMTs的底物特异性的肽阵列。对于酶modifi阳离子，肽阵列一起温育的各PKMT和标记的[甲基- 3 H] - -AdoMet在适当的缓冲液中。各个基板的甲基化是由以下的放射性标记的甲基的距离的AdoMet通过放射自显影（ 图3）的酶促转移到肽底物进行分析。通过此过程中，肽阵列允许不同的肽底物的甲基化的同时研究。这种方法的一个重要优点是，所有的肽都甲基化的竞争，使得在所述甲基化动力学的线性相位，每种肽的相对甲基化正比于催化速率常数通过解离常数（k 猫 / K除以D中的酶用于各个肽底物）。因此，放射性掺入每个斑点的量直接与朝向特定的肽酶活性。使用的肽阵列甲基实验的结果来看，PKMT的特异性轮廓可以定义和基于这一新基材可为前提。肽阵列允许的新颖底物在肽水平的甲基化的快速和成本有效的验证。对于这一点，阵列是制备包含该预测的新颖底物与含有丙氨酸代替赖氨酸在靶位点修饰的肽以及阳性和阴性对照的肽一起。最后，新的衬底可以被制备为蛋白质连同突变体，其中靶的Lys被改变为丙氨酸和甲基化可以在蛋白质水平证实。根据结果​​，这随后是生物学研究解决新描述蛋白质底物的甲基化的潜在作用。

<table border="0" cellpadding="0" cellspacing="0" width="734">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Name of Material/ Equipment</td>
			<td style="width:112px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:179px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol abs Gradient Grade HPLC</td>
			<td>Honeywell</td>
			<td>10299901</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N-Dimethylformamide Peptide Synthesis</td>
			<td>Biosolve</td>
			<td style="width:119px;">4193302</td>
			<td>Flammable, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Piperidine &#8805; 99 % for Peptide Synthesis</td>
			<td>Roth</td>
			<td>A122.1</td>
			<td>Flammable, Toxic, corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Oxyma Pure (Ethyl (hydroxyimino)cyanoacetate )</td>
			<td>Novabiochem</td>
			<td>8510860100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">N,N&#8217;-Diisopropylcarbodiimide purum &#8805; 98% GC</td>
			<td>Fluka</td>
			<td>38370</td>
			<td>Flammable, Toxic, corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic anhydride</td>
			<td>Roth</td>
			<td>CP28.1</td>
			<td>Flammable, Toxic, corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triisopropylsilane 99%,</td>
			<td>Aldrich</td>
			<td>233781</td>
			<td>Flammable, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dichlormethane &#8805;99,9%</td>
			<td>Roth</td>
			<td>P089.1</td>
			<td>Carcinogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Methyl-2-Pyrrolidone</td>
			<td>Roth</td>
			<td>4306.2</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Derivatized cellulose Membrane</td>
			<td style="width:112px;">Intavis AG K&#246;ln</td>
			<td style="width:119px;">32.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trifluoroacetic acid</td>
			<td>Roth</td>
			<td>P088.2</td>
			<td>Toxic, corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromphenolblue</td>
			<td>AppliChem</td>
			<td>A3640.0010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Hydrogen Carbonate</td>
			<td>Roth</td>
			<td>T871.2</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Dodecyl Sulfate Pellets</td>
			<td>Roth</td>
			<td>CN30.3</td>
			<td>Toxic, Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">MultiPep Synthesizer</td>
			<td style="width:112px;">Intavis AG K&#246;ln</td>
			<td style="width:119px;">n.a.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">HyperfilmTM high performance film</td>
			<td style="width:112px;">GE Healthcare</td>
			<td style="width:119px;">28906837</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Phoretix software</td>
			<td style="width:112px;">TotalLab</td>
			<td style="width:119px;">n.a.</td>
			<td style="width:179px;"></td>
		</tr>
	</tbody>
</table>

specificity analysis of protein lysine methyltransferases using spot peptide arrays

赖氨酸的甲基化是一个新兴的翻译后修饰，这已被确定在几个组蛋白和非组蛋白，它在细胞发育和许多疾病的关键角色。大约5000赖氨酸甲基化位点被确定在不同的蛋白质，这是由数十个蛋白赖氨酸甲基转移设置。这表明，每PKMT甲基化多种蛋白，但至今只有一个或两个基片已被确定为若干这些酶。 PKMTs来解决这个问题，我们引入肽基于阵列的底物特异性分析。肽阵列功能强大的工具来PKMTs的特异性表征，因为几个底材具有不同序列的甲基化可以在一个阵列进行测试。我们使用Intavis SPOT合成仪合成肽阵列纤维素膜和分析各种PKMTs的特异性。基于该结果，对于几个这些酶的，新的substrates可以被识别。例如，对于NSD1通过采用肽阵列，我们表明，甲基化H4的K44代替报告H4K20并且另外H1.5K168高于先前已知的H3K36高度优选的基材。因此，肽阵列功能强大的工具，生化表征PKMTs。

肽阵列被成功地用于生化表征PKMTs的特异性，和几个新的组蛋白和非组蛋白PKMT的衬底被确定通过该方法5,16-19。定义PKMT（或任何酶）的正确基材频谱是迈向它的分子机制和细胞功能的理解的重要一步。 作为肽的SPOT阵列甲基方法的应用的一个例子，我们描述了NSD1 PKMT 19的特异性分析的结果。以前的我们的工作，这种酶被描述为甲醇几个基材，包括组蛋白H3的K36?...

Watch this Scientific Journal Video about Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays at JoVE.com

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

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 ...

specificity-analysis-protein-lysine-methyltransferases-using-spot

Research

JoVE Journal

Biology

13.8K 次观看.  Stuttgart University. 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.

视频: Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

加上质谱肽免疫亲和富集的蛋白质定量

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

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 molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

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 ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

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 ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

表面拴DNA大分子的可视化与荧光蛋白的DNA结合肽

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...