A Proteomic Approach to Biomarker Discovery in Prion Disease

描述(由申请人提供)：传染性海绵状脑病(TSE)是一种独特的中枢神经系统退行性疾病家族，通常是致命的。在各种形式的Pron疾病中，对人和动物健康影响最大的是CreutzFeldt-Jakob病(CJD)、牛海绵状脑病(BSE)、瘙痒病和慢性衰减病(CWD)。疯牛病和变种CJD(VCJD)在英国和包括美国在内的世界范围内的暴发，促使人们需要快速、可靠和廉价的筛查方法来检测人和活动物中的TSE感染。到目前为止，目前对Prion病的诊断测试主要集中在检测疾病的病因、异常的Prion蛋白或与神经系统疾病相关的个别蛋白。这些检测是不充分的，因为它们是死后检测，吞吐量低，而且不够敏感，无法在临床前期早期发现感染。显然，迫切需要开发一种可靠、灵敏和特异的死前诊断试验，用于临床前识别感染TSE的动物或个人。在此，我们建议开发一种新的基于高灵敏质谱学(MS)的分析平台，并结合一套生物信息学工具来识别从临床前潜伏期感染PrP疾病的活体动物身上收集的一组Pron疾病的生物标志物。这项研究的指导假设是：(1)与Pron感染相关的病理生理变化将导致脑脊液(CSF)和/或血清蛋白质组的蛋白质谱改变。(2)准确的质量测量、定量分析的同位素标记策略、串联质谱仪和一套机器学习算法的组合将提供一个独特的集成平台，以前所未有的灵敏度和特异性识别和表征这些诊断蛋白质标记物。(3)这些诊断蛋白生物标志物的特征将为发展基于抗体的可疑动物普恩疾病的生前筛查试验提供基础。为了研究这些假说，我们提出了以下具体目标：(1)利用MS和分类算法在感染Pron疾病的动物的脑脊液和血清中识别一组候选生物标志物。(2)利用串联质谱仪对候选生物标志物进行鉴定，并通过同位素标记获得可能生物标志物的整体定量变化。(3)生物标志物在盲法诊断试验中的验证。总而言之，这项拟议的研究将为在死前样本中快速而灵敏地诊断普恩病毒病提供基础，并将进一步加深我们对普恩病毒疾病进展和病理的理解。这项建议探索了一种新的蛋白质组学方法，将高灵敏度的质谱分析和测序与一套生物信息学工具相结合，以发现、识别和量化指示普恩病毒感染的生物标记物。这些假定的生物标志物也可以作为治疗干预的靶点。总的来说，拟议的研究将为在死前样本中开发快速和灵敏的普恩病毒疾病诊断方法提供基础，增强我们确保食物和血液供应的能力，并将进一步加深我们对普恩病毒疾病进展和病理的了解。

DESCRIPTION (provided by applicant): Transmissible spongiform encephalopathies (TSEs) are a unique family of neurodegenerative diseases of the central nervous system that are always fatal. Of the various forms of prion disease, those with the most impact upon human and animal health are Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), scrapie and chronic wasting disease (CWD). The outbreaks of BSE and variant CJD (vCJD) in the United Kingdom and worldwide, including the United States, has prompted the need for rapid, reliable and inexpensive screening methods that detect TSE infection in humans and live animals. Current diagnostic tests for prion disease have, thus far, focused on detection of the causal agent of the disease, the abnormal prion protein, or individual proteins that correlate with the neurological disease. These tests are inadequate because they are post-mortem, low throughput and not sufficiently sensitive to detect infection early in the pre-clinical period. Clearly, there is an urgent need for the development of a reliable, sensitive, and specific ante-mortem diagnostic test for the pre-clinical identification of TSE-infected animals or individuals. Herein we propose the development of a new highly-sensitive mass spectrometry (MS)-based analytical platform in combination with a suite of bioinformatics tools to identify a panel of biomarkers of prion disease in body fluids collected from live animals infected with prion disease during the preclinical incubation period. The guiding hypotheses of the proposed research are: (1) The pathophysiological changes associated with prion infection will result in an altered protein profile of the cerebrospinal fluids (CSF) and/or serum proteome. (2) The combination of accurate mass measurements, isotopic labeling strategy for quantitative analyses, tandem mass spectrometry, and a suite of machine-learning algorithms will provide a unique and integrated platform to identify and characterize these diagnostic protein markers with unprecedented sensitivity and specificity. (3) Characterization of these diagnostic protein biomarkers will provide a basis for the development of an ante-mortem antibody-based screening test for prion diseases in suspected animals. To investigate these hypotheses, we propose the following specific aims: (1) To identify a panel of candidate biomarkers in the CSF and serum of animals infected with prion diseases using MS and classification algorithms. (2) To determine the identities of the candidate biomarkers by tandem MS and to obtain global quantitative changes of putative biomarkers via isotopic labeling. (3) To validate the biomarkers in blind diagnostic trials. Collectively, the proposed study will provide a basis for the development of a rapid and sensitive diagnosis of prion disease in an ante-mortem sample and it will also further our understanding of prion disease progression and pathology. This proposal explores a novel proteomic approach combining high sensitivity mass spectrometric profiling and sequencing with a set of bioinformatics tools to discover, identify, and quantify biomarkers that are indicative of prion infection. These putative biomarkers may also serve as targets for therapeutic intervention. Collectively, the proposed research will provide a basis for the development of a rapid and sensitive diagnosis of prion disease in an ante-mortem sample, enhancing our ability to secure food and blood supplies, and it will also further our understanding of prion disease progression and pathology.