Multiplexed In Solution Protein Array (MISPA) for identifying novel protein interactions in cancer and for early detection of immune responses in pathogen-induced cancers

多重溶液蛋白阵列 (MISPA) 用于识别癌症中的新型蛋白质相互作用以及早期检测病原体诱导的癌症中的免疫反应

• 批准号: 8929424
• 负责人: JOSHUA LABAER
• 金额: $ 23.52万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-15 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8929424
• 关键词: Address; Affinity Chromatography; Antigens; B lymphoid malignancy; Binding; Binding Proteins; Biological Assay; Cancer Biology; Cancer Diagnostics; Clinical; Clinical Management; Consumption; DNA; DNA Sequence; Detection; Development; Diagnostic; Disease; Disease Marker; Equipment; Event; Fluorescence; Genes; Human; Human Papillomavirus; Human papilloma virus infection; Immune response; Incubated; Individual; Kinetics; Lead; Libraries; Link; Liquid substance; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Methods; Nature; Nucleic Acids; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Populations at Risk; Production; Protein Array; Protein Microchips; Proteins; Proteome; Receptors, Antigen, B-Cell; Research; Running; Sampling; Screening for cancer; Serotyping; Serum; Solutions; Surface; System; Testing; Time; base; cDNA Library; cancer therapy; clinical material; flexibility; genetic approach; high throughput screening; innovation; macromolecule; malignant oropharynx neoplasm; member; next generation; next generation sequencing; novel; pathogen; programs; protein complex; protein folding; protein profiling; protein protein interaction; public health relevance; research study; screening; skills; targeted cancer therapy; therapy development; tool; yeast two hybrid system

 DESCRIPTION (provided by applicant): The measurement of protein-protein interactions (PPI) drives both our understanding of cancer development and routinely contributes to its clinical management. Both discovery and diagnostics increasingly rely on multiplexed assays for PPI. Protein microarrays provide a powerful approach to identify PPI efficiently in high- throughput. They allow proteome-scale screening with low sample consumption and are compatible with testing clinical material. With support from IMAT, we developed an innovative method for producing protein microarrays called nucleic acid programmable protein arrays (NAPPA) that produces properly folded proteins in a human milieu with consistent yields, "just-in-time" for the assay. NAPPA has proven to be an accurate and flexible tool for studying both novel PPI and patient immune responses. However, like all protein microarrays, NAPPA is subject to binding kinetics of surface-bound proteins, non-specific background binding and the limited dynamic range of fluorescence. Here, we propose the development of a next-generation, liquid-phase protein microarray platform, "Multiplex In Solution Protein Array" (MISPA), which exploits the extraordinary dynamic range of nucleic acid measurement and its wide availability in both research and clinical labs. We have developed a unique method to produce and "barcode" individual proteins, which can then interact in solution with a test molecule or clinical sample to separate targets from background. The barcodes are amplified and then assessed quantitatively by next generation sequencing (NGS) or qPCR. Because separate experiments can themselves be barcoded, multiple experiments can be combined and all the results determined in a single NGS run. We will test the feasibility of MISPA by investigating protein-protein interactions in the B-cell receptor (BCR) pathway, which is a prime target for cancer therapy and responsible for many B-cell malignancies. It provides an excellent test system because it includes both well-studied interactions and many unknown interactions awaiting exploration. We will probe 100 members of the BCR pathway with 10 key query proteins and analyze their interactions by NGS. We also propose to implement a robust, multiplexed qPCR-based diagnostic tool for studying immune responses in pathogen-induced cancers. We will focus on oropharyngeal carcinomas (OPC), which have been linked to human papillomavirus (HPV) infection. The proteomes of 10 different serotypes of HPV will be barcoded and tested against HPV positive OPC serum samples and control samples (N=20 each). This method expands on the key innovations of our NAPPA platform that included, programmability to test virtually any protein, in human milieu to encourage function, just-in-time protein production and equimolar protein display, and will also include new innovations including solution-phase binding kinetics, expanded dynamic range, compatibility with clinical diagnostics, reduced non-specific background, and multiplexed experimentation.

 描述（由申请人提供）：蛋白质-蛋白质相互作用（PPI）的测量推动了我们对癌症发展的理解，并对其临床管理做出了常规贡献。发现和诊断都越来越依赖于PPI的多重测定。蛋白质微阵列技术为高通量、高效率地鉴定PPI提供了一种强有力的方法.它们允许以低样品消耗进行蛋白质组规模的筛选，并且与测试临床材料兼容。在IMAT的支持下，我们开发了一种用于生产蛋白质微阵列的创新方法，称为核酸可编程蛋白质阵列（NAPPA），该方法可以在人类环境中以一致的产量生产正确折叠的蛋白质，“及时”用于测定。NAPPA已被证明是研究新型PPI和患者免疫反应的准确和灵活的工具。然而，像所有的蛋白质微阵列，NAPPA是受表面结合的蛋白质，非特异性背景结合和有限的动态范围的荧光结合动力学。在这里，我们建议开发下一代液相蛋白质微阵列平台，“多重溶液蛋白质阵列”（MISPA），它利用了核酸测量的非凡动态范围及其在研究和临床实验室中的广泛可用性。我们已经开发出一种独特的方法来生产和“条形码”个别蛋白质，然后可以在溶液中与测试分子或临床样品相互作用， 将目标与背景分开。扩增条形码，然后通过下一代测序（NGS）或qPCR定量评估。由于单独的实验本身可以被条形码化，因此可以组合多个实验，并且在单个NGS运行中确定所有结果。我们将通过研究蛋白质-蛋白质相互作用来测试MISPA的可行性。 B细胞受体（BCR）通路，是癌症治疗的主要靶点，并导致许多B细胞恶性肿瘤。它提供了一个很好的测试系统，因为它既包含了研究得很好的交互作用，也包含了许多未知的交互作用。我们将用10个关键查询蛋白探测BCR途径的100个成员，并通过NGS分析它们的相互作用。我们还建议实施一个强大的，基于多重qPCR的诊断工具，用于研究病原体诱导的癌症中的免疫反应。我们将重点关注口咽癌（OPC），这与人乳头瘤病毒（HPV）感染有关。将对10种不同血清型HPV的蛋白质组进行条形码化，并针对HPV阳性OPC血清样本和对照样本（各N=20）进行检测。这种方法扩展了我们NAPPA平台的关键创新，包括可编程性，以测试几乎任何蛋白质，在人类环境中促进功能，及时蛋白质生产和等摩尔蛋白质展示，还将包括新的创新，包括溶液相结合动力学，扩大动态范围，与临床诊断的兼容性，减少非特异性背景和多重实验。