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

用于癌症相关翻译后修饰的高密度肽阵列

基本信息

批准号：
8738628
负责人：
Mary Szatkowski Ozers
金额：
$ 20.78万
依托单位：
PROTEOVISTA, LLC
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-20 至 2016-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8738628
关键词：
Address Affinity Amino Acids Antibodies Arginine Biological Assay Biological Markers Bypass Cancer Etiology Cancer Patient Cancer cell line Clinical Code Complex Cysteine DNA DNA Microarray Chip DNA Sequence DNA biosynthesis Data Detection Development Devices Diagnostic Diagnostic Neoplasm Staging Drug Targeting Ensure Enzymes Epitopes Equipment Future Gene Expression Genetic Transcription Glass Grant Human Image In Vitro Indolent Label Lead Location Malignant Neoplasms Maps Mass Spectrum Analysis Methods Methylation Microarray Analysis Nature Patients Pattern Peptides Phage Display Phosphorylation Phosphotransferases Play Post Translational Modification Analysis Post-Translational Modification Site Post-Translational Protein Processing Procedures Process Protein Conformation Proteins Proteome Proteomics RNA Regimen Research Personnel Role S-nitro-N-acetylpenicillamine Sampling Series Signal Transduction Slide Specificity Spottings Surface Technology Therapeutic Time Translations Tyrosine Phosphorylation anticancer research arginine methyltransferase cancer cell cancer type cellular targeting cost density design direct application enzyme activity enzyme substrate genome-wide innovation interest novel point of care point-of-care diagnostics protein aminoacid sequence protein protein interaction public health relevance research study screening small molecule success therapeutic development tumor progression

项目摘要

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.

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，酶促的蛋白质相互作用在癌症病因和进展中起关键作用活性和蛋白质构象。肽阵列在与癌症相关的发现中发挥了重要作用，例如癌症生物标志物，护理点诊断和针对蛋白质 - 蛋白质相互作用的治疗剂。该应用程序将开发一种集成技术，即快照阵列（对肽的特异性和亲和力），以使用转录和翻译机械在单个载玻片上合成一百万个独特的肽。创新的快照阵列的完整性将在三个步骤的过程中进行验证，涉及质谱法，氨基酸的体外荧光标记以及抗体识别，以确保在此新过程中准确合成肽。然后，将通过纯化的酶来修饰快照阵列上的肽，这些酶赋予磷酸化，sumoylation和精氨酸甲基化的常见癌症相关的翻译后修饰。从癌细胞制备的裂解物将应用于快照阵列，以评估其在阵列肽上赋予翻译后修饰的能力的差异。该赠款的具体目的是：1。开发和验证一种体外方法，以合成在人蛋白质组中显示所有肽的高密度肽微阵列。 2。通过使用纯化的酶赋予磷酸化，sumoylation和甲基化标记，修饰阵列上的肽。 3。定量两种癌细胞系之间酪氨酸磷酸化活性的差异。当前可用的光刻或斑点肽阵列显示100-10,000个肽，而快照策略将在单个载玻片上合成一百万个独特的肽，提供更大的吞吐量。快照阵列将以标准DNA微阵列的成本（每个阵列<$ 1000）开发，该阵列的成本大大低于当前肽阵列的成本，仅显示了人类蛋白质组的一小部分。快照阵列是由一系列仔细的时序和精确的步骤合成的，该阵列的体外DNA复制，RNA转录和肽翻译。一旦合成，将肽在新的过程中重新连接到载玻片上的特定地址，并且在低体积室内的载玻片上可以发生任何实验。这种设计极大地简化了肽阵列实验，并避免了对昂贵的设备或复杂程序（例如质谱或噬菌体显示）的需求。快照阵列的直接应用包括对关键蛋白药物靶标的小分子筛选，新型癌症生物标志物的鉴定以及护理点诊断设备的开发，以评估癌症患者的生物测量。