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

 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，并对其临床治疗做出了常规贡献。发现和诊断都越来越依赖于PPI的多路分析。蛋白质芯片为高通量高效鉴定PPI提供了一种强有力的方法。它们允许以低样本消耗进行蛋白质组规模的筛选，并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)，它在人类环境中以稳定的产量产生正确折叠的蛋白质，用于分析。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一样，NAPPA受制于表面结合蛋白的结合动力学、非特定背景结合和有限的荧光动态范围。在这里，我们建议开发下一代液态蛋白质微阵列平台--“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”单个蛋白质，然后可以在溶液中与测试分子或临床样本相互作用，以 将目标与背景分开。条形码被扩增，然后通过下一代测序(NGS)或qPCR进行定量评估。因为单独的实验本身可以是条形码的，所以多个实验可以组合在一起，所有结果都可以在一次NGS运行中确定。我们将通过研究蛋白质与蛋白质之间的相互作用来测试MISPA的可行性 B细胞受体(BCR)途径，是癌症治疗的主要靶点，与许多B细胞恶性肿瘤有关。它提供了一个很好的测试系统，因为它既包括研究得很好的相互作用，也包括许多有待探索的未知相互作用。我们将用10个关键的查询蛋白来探测100个BCR通路成员，并用NGS分析它们之间的相互作用。我们还建议实现一个强大的，基于多路qPCR的诊断工具，用于研究病原体诱导的癌症的免疫反应。我们将关注口咽癌(OPC)，它与人类乳头瘤病毒(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编码，并与HPV阳性的OPC血清样本和对照样本(N=20)进行检测。该方法扩展了我们的NAPPA平台的关键创新，包括在人体环境中测试几乎任何蛋白质的可编程性，以鼓励功能、即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、扩大的动态范围、与临床诊断的兼容性、减少非特异性背景和多路实验。

 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.