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。在各种形式的朊病毒疾病中，对人类和动物健康影响最大的是克雅氏病（CJD）、牛海绵状脑病（BSE）、痒病和慢性消耗性疾病（CWD）。在英国和包括美国在内的世界范围内，疯牛病和变异型克雅氏病（vCJD）的暴发促使人们需要快速、可靠和廉价的筛查方法，以检测人类和活体动物中的TSE感染。迄今为止，目前对朊病毒疾病的诊断测试主要集中于检测疾病的致病因子、异常朊病毒蛋白或与神经系统疾病相关的单个蛋白质。这些检测是不充分的，因为它们是死后检测，低通量，而且不够敏感，无法在临床前早期发现感染。显然，迫切需要开发一种可靠、敏感和特异性的死前诊断测试，用于临床前鉴定感染tse的动物或个体。在此，我们建议开发一种新的基于高灵敏度质谱（MS）的分析平台，结合一套生物信息学工具，在临床前潜伏期感染了朊病毒疾病的活体动物的体液中识别一组朊病毒疾病的生物标志物。提出的研究的指导假设是：(1)与朊病毒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)精确的质量测量、定量分析的同位素标记策略、串联质谱和一套机器学习算法的结合，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性蛋白质标记物。(3)这些诊断性蛋白生物标志物的鉴定将为在疑似动物中开发基于抗体的死前朊病毒疾病筛选试验提供基础。为了验证这些假设，我们提出了以下具体目标：(1)使用质谱和分类算法在感染朊病毒疾病的动物CSF和血清中识别一组候选生物标志物。(2)通过串联质谱法确定候选生物标志物的身份，并通过同位素标记获得推定生物标志物的全局定量变化。(3)在盲诊断试验中验证生物标志物。总的来说，这项研究将为在死前样本中快速、灵敏地诊断朊病毒疾病提供基础，也将进一步加深我们对朊病毒疾病进展和病理的理解。本研究探索了一种新的蛋白质组学方法，将高灵敏度质谱分析和测序与一套生物信息学工具相结合，以发现、鉴定和量化指示朊病毒感染的生物标志物。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快速和敏感地诊断朊病毒疾病提供基础，增强我们确保食物和血液供应的能力，也将进一步加深我们对朊病毒疾病进展和病理的理解。

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.