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><tbody><tr><td>Ethanol abs Gradient Grade HPLC</td><td>Honeywell</td><td>10299901</td><td>Flammable</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide Peptide Synthesis</td><td>Biosolve</td><td>4193302</td><td>Flammable, Toxic</td></tr><tr><td>Piperidine &amp;ge;99% for Peptide Synthesis</td><td>Roth</td><td>A122.1</td><td>Flammable, Toxic, corrosive</td></tr><tr><td>Oxyma Pure (Ethyl (hydroxyimino)cyanoacetate )</td><td>Novabiochem</td><td>8510860100</td><td></td></tr><tr><td>&lt;em&gt;N,N&amp;rsquo;&lt;/em&gt;-Diisopropylcarbodiimide purum &amp;ge;98% GC</td><td>Fluka</td><td>38370</td><td>Flammable, Toxic, corrosive</td></tr><tr><td>Acetic anhydride</td><td>Roth</td><td>CP28.1</td><td>Flammable, Toxic, corrosive</td></tr><tr><td>Triisopropylsilane 99%</td><td>Aldrich</td><td>233781</td><td>Flammable, Toxic</td></tr><tr><td>Dichlormethane &amp;ge;99.9%</td><td>Roth</td><td>P089.1</td><td>Carcinogenic</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-Methyl-2-Pyrrolidone</td><td>Roth</td><td>4306.2</td><td>Toxic</td></tr><tr><td>Derivatized cellulose Membrane</td><td>Intavis AG K&amp;ouml;ln</td><td>32.1</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Roth</td><td>P088.2</td><td>Toxic, corrosive</td></tr><tr><td>Bromphenolblue</td><td>AppliChem</td><td>A3640.0010</td><td></td></tr><tr><td>Ammonium Hydrogen Carbonate</td><td>Roth</td><td>T871.2</td><td>Toxic</td></tr><tr><td>Sodium Dodecyl Sulfate Pellets</td><td>Roth</td><td>CN30.3</td><td>Toxic, Flammable</td></tr><tr><td>MultiPep Synthesizer</td><td>Intavis AG K&amp;ouml;ln</td><td>n.a.</td><td></td></tr><tr><td>HyperfilmTM high performance film</td><td>GE Healthcare</td><td>28906837</td><td></td></tr><tr><td>Phoretix software</td><td>TotalLab</td><td>n.a.</td><td></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

13.9K 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

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

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important for revealing protease function. The method proposed in this study determines protease specificity by measuring the molecular weight of unique substrates using Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The substrates contain iminobiotin, while the cleaved site consists of amino acids, and the spacer consists of polyethylene glycol. The cleaved substrate will generate a unique molecular weight using a cleaved amino acid. One of the merits of this method is that it may be carried out in one pot using crude samples, and it is also suitable for assessing multiple samples. In this article, we describe a simple experimental method optimized with samples extracted from mouse lung tissue, including tissue extraction, placement of digestive substrates into samples, purification of digestive substrates under different pH conditions, and measurement of the substrates&#39; molecular weight using MALDI-TOF mass spectrometry. In summary, this technique allows for the identification of protease specificity in crude samples derived from tissue extracts using MALDI-TOF mass spectrometry, which may easily be scaled up for multiple sample processing.

Here, we present a protocol to determine protease specificity in crude mouse tissue extracts using MALDI-TOF spectrometry.

MALDI 质谱法测定小鼠组织提取物蛋白酶特异性: 操作 PH 值导致特异性改变

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

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

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

基于质谱的蛋白质组学方法用于全局和高置信度的蛋白质R-甲基化分析

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized. Peptidomic studies frequently analyze peptides that are naturally generated in cells. Here, a sample preparation protocol based on heat-inactivation is described, which eliminates protease activity, and extraction with mild conditions, so there is no peptide bonds cleavage. In addition, the relative quantitation of peptides using stable isotope labeling by reductive methylation of amines is also shown. This labeling method has some advantages as the reagents are commercially available, inexpensive compared to others, chemically stable, and allows the analysis of up to five samples in a single LC-MS run.

This article describes a sample preparation method based on heat-inactivation to preserve endogenous peptides avoiding degradation post-mortem, followed by relative quantitation using isotopic labeling plus LC-MS.

使用胺还原甲基化进行样品制备和相对定量，用于肽学研究

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the ...

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 ...

生物学

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 ...

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to human brain function, as neurons are postmitotic and rely heavily on the autophagy-lysosome pathway to maintain cellular homeostasis. Despite advancements in the understanding of various lysosomal functions, capturing the highly dynamic communications between lysosomes and other cellular components is technically challenging, particularly in a high-throughput fashion. Here, a detailed protocol is provided for the recently published endogenous (knock-in) lysosome proximity labeling proteomic method in human induced pluripotent stem cell (hiPSC)-derived neurons. 
Both lysosomal membrane proteins and proteins surrounding lysosomes within a 10-20 nm radius can be confidently identified and accurately quantified in live human neurons. Each step of the protocol is described in detail, i.e., hiPSC-neuron culture, proximity labeling, neuron harvest, fluorescence microscopy, biotinylated protein enrichment, protein digestion, LC-MS analysis, and data analysis. In summary, this unique endogenous lysosomal proximity labeling proteomics method provides a high-throughput and robust analytical tool to study the highly dynamic lysosomal activities in live human neurons.

A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

具有邻近标记蛋白质组学的神经元溶酶体相互作用组表征

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to ...

神经科学

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 ...

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The &#34;one-pot&#34; affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

量化和站点本地化两个翻译后修改的同步亲和力丰富

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into ...