High Density Peptide Arrays for Cancer-Related Post-Translational Modifications

描述（由申请人提供）：蛋白质的翻译后修饰通过改变蛋白质-蛋白质相互作用、酶促作用和蛋白质-蛋白质相互作用，在癌症病因学和进展中发挥关键作用。 活性和蛋白质构象。肽阵列在癌症相关的发现中发挥了重要作用，例如癌症生物标志物，即时诊断和针对蛋白质-蛋白质相互作用的治疗。该申请将开发一种集成技术，SNAP-Tide阵列（PepTides的特异性和亲和力），使用转录和翻译机制在单个载玻片上合成100万个独特的肽。创新SNAP-Tide阵列的完整性将在三步过程中得到验证，包括质谱法，氨基酸的体外荧光标记和抗体识别，以确保在这种新工艺中准确合成肽。然后，SNAP-Tide阵列上的肽将被纯化的酶修饰，所述纯化的酶赋予磷酸化、类小泛素化和精氨酸甲基化的常见癌症相关的翻译后修饰。将从癌细胞制备的裂解物应用于SNAP-Tide阵列，以评估它们赋予阵列肽翻译后修饰的能力的差异。该补助金的具体目标是：1。开发并验证一种体外合成高密度肽微阵列的方法，以显示人类蛋白质组中的所有肽。2.通过使用纯化的酶赋予磷酸化、类小泛素化和甲基化标记来修饰阵列上的肽。3.两种癌细胞系之间酪氨酸磷酸化活性的定量差异。目前可用的平版印刷或点样肽阵列显示100- 10，000种肽，而SNAP-Tide策略将在单个载玻片上合成100万种独特的肽，提供更大的通量。SNAP-Tide阵列将以标准DNA微阵列的成本开发（每个阵列<1000美元），这明显低于目前仅显示一部分人类蛋白质组的肽阵列。SNAP-Tide阵列是通过一系列精心定时和精确的体外DNA复制、RNA转录和肽翻译步骤合成的。一旦合成，肽在一个新的过程中重新连接到载玻片上的特定地址，并且任何实验都可以在小体积腔室内的载玻片上进行。这种设计大大简化了肽阵列实验，并避免了对昂贵设备或复杂程序的需要，如质谱或噬菌体展示。SNAP-Tide阵列的直接应用包括关键蛋白质药物靶点的小分子筛选、新型癌症生物标志物的鉴定以及用于评估癌症患者生物标本的即时诊断设备的开发。

DESCRIPTION (provided by applicant): Post-translational modifications of proteins play a pivotal role in cancer etiology and progression by altering protein-protein interactions, enzymatic activity, and protein conformation. Peptide arrays have played a significant role in cancer-related discoveries, such as cancer biomarkers, point-of-care diagnostics, and therapeutics directed at protein-protein interactions. This application will develop an integrated technology, the SNAP-Tide array (Specificity and Affinity for PepTides), to synthesize one million unique peptides on a single glass slide using transcriptional and translational machinery. The integrity of the innovative SNAP-Tide arrays will be validated in a three step process, involving mass spectrometry, in vitro fluorescent labeling of amino acids, and antibody recognition to ensure the peptides are accurately synthesized in this novel process. The peptides on the SNAP-Tide array will then be modified by purified enzymes that confer the common cancer- related post-translational modifications of phosphorylation, sumoylation, and arginine methylation. Lysates prepared from cancer cells will be applied to the SNAP-Tide array to evaluate differences in their ability to confer post-translational modifications on the array peptides. The specific aims o this grant are to: 1. Develop and validate an in vitro method to synthesize high density peptide microarrays that display all peptides in the human proteome. 2. Modify the peptides on the array by conferring phosphorylation, sumoylation, and methylation marks using purified enzymes. 3. Quantitate differences in tyrosine phosphorylation activity between two cancer cell lines. Currently available lithographic or spotted peptide arrays display 100-10,000 peptides, while the SNAP-Tide strategy will synthesize a million unique peptides on a single glass slide offering substantially greater throughput. The SNAP-Tide array will be developed for the cost of a standard DNA microarray (<$1000 per array), which is significantly less than current peptide arrays displaying only a fraction of the human proteome. The SNAP-Tide array is synthesized by a series of carefully timed and precise steps of in vitro DNA replication, RNA transcription, and peptide translation. Once synthesized, the peptides are re-attached in a novel process to specific addresses on the glass slide, and any experiments can occur on the glass slide within the low- volume chamber. This design greatly simplifies peptide array experiments and avoids the need for expensive equipment or complicated procedures, such as mass spectrometry or phage display. Direct applications of the SNAP-Tide array include small molecule screening of pivotal protein drug targets, identification of novel cancer biomarkers, and development of point-of-care diagnostic devices to evaluate cancer patient biospecimens.