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

描述(申请人提供)：蛋白质的翻译后修饰通过改变蛋白质-蛋白质之间的相互作用，酶促，在癌症的病因和进展中发挥关键作用 活性和蛋白质构象。多肽阵列在癌症相关发现中发挥了重要作用，如癌症生物标记物、护理点诊断和针对蛋白质-蛋白质相互作用的治疗。这项应用将开发一种集成技术，SNAP-Tide阵列(多肽的特异性和亲和力)，使用转录和翻译机制在一张玻璃片上合成100万个独特的多肽。创新的SNAP-Tide阵列的完整性将通过三个步骤进行验证，包括质谱学、氨基酸的体外荧光标记和抗体识别，以确保在这一新工艺中准确地合成多肽。SNAP-Tide阵列上的多肽随后将被纯化的酶修饰，这些酶实现了常见的癌症相关的翻译后修饰，如磷酸化、SUMO化和精氨酸甲基化。从癌细胞制备的裂解物将被应用于SNAP-Tide阵列，以评估它们对阵列多肽进行翻译后修饰的能力的差异。这项资助的具体目的是：1.开发和验证一种体外合成高密度多肽芯片的方法，该芯片可以显示人类蛋白质组中的所有多肽。2.通过使用纯化的酶赋予磷酸化、苏莫化和甲基化标记来修饰阵列上的多肽。3.定量测定两种癌细胞系之间酪氨酸磷酸化活性的差异。目前可用的平版或斑点多肽阵列可显示100-10,000个多肽，而SNAP-Tide策略将在一张玻璃片上合成100万个独特的多肽，提供显著更大的吞吐量。SNAP-Tide阵列的开发成本相当于标准DNA微阵列(每个阵列1000美元)，这比目前只显示一小部分人类蛋白质组的多肽阵列要少得多。SNAP-Tide阵列是通过一系列精心计时和精确的体外DNA复制、RNA转录和多肽翻译步骤合成的。一旦合成，这些肽就会以一种新的工艺重新连接到玻片上的特定地址，任何实验都可以在低容量腔体内的玻片上进行。这种设计极大地简化了肽阵列实验，并避免了昂贵的设备或复杂的程序，如质谱仪或噬菌体展示。SNAP-Tide阵列的直接应用包括关键蛋白质药物靶标的小分子筛选、新的癌症生物标记物的识别以及用于评估癌症患者生物样本的护理点诊断设备的开发。

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.