A Proteomic Approach to Biomarker Discovery in Prion Disease

描述（由申请方提供）：传染性海绵状脑病（TSE）是一种独特的中枢神经系统神经退行性疾病家族，通常具有致死性。在各种形式的朊病毒病中，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）、羊瘙痒病和慢性消耗病（CWD）。BSE和变异型CJD（vCJD）在英国和包括美国在内的世界范围内的爆发，促使人们需要快速、可靠和廉价的筛查方法来检测人类和活动物中的TSE感染。目前，朊病毒病的诊断测试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。这些测试是不充分的，因为它们是死后的，低通量的，并且在临床前阶段的早期检测感染的灵敏度不够。显然，迫切需要开发一种可靠、灵敏和特异的死前诊断试验，用于临床前鉴定TSE感染的动物或个体。在此，我们提出了一个新的高灵敏度的质谱（MS）为基础的分析平台，结合一套生物信息学工具，以确定一组生物标志物的朊病毒疾病的体液中收集的活动物感染的朊病毒疾病在临床前潜伏期。本研究的指导性假设是：（1）朊病毒感染引起的病理生理学改变将导致脑脊液（CSF）和/或血清蛋白质组的蛋白质谱改变。(2)精确的质量测量、用于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组合将提供一个独特的综合平台，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)这些诊断性蛋白质生物标志物的表征将为在疑似动物中开发基于死前抗体的朊病毒疾病筛查试验提供基础。为了研究这些假设，我们提出了以下具体目标：（1）使用MS和分类算法在感染朊病毒疾病的动物的CSF和血清中鉴定一组候选生物标志物。(2)通过串联质谱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总体定量变化。(3)在盲法诊断试验中验证生物标志物。总的来说，拟议中的研究将提供一个基础的发展，一个快速和敏感的诊断朊病毒疾病的死前样本，它也将进一步了解朊病毒疾病的进展和病理。该提案探索了一种新的蛋白质组学方法，该方法将高灵敏度质谱分析和测序与一组生物信息学工具相结合，以发现，鉴定和定量指示朊病毒感染的生物标志物。这些推定的生物标志物也可以作为治疗干预的靶点。总的来说，拟议中的研究将为在死前样本中开发朊病毒疾病的快速和灵敏的诊断提供基础，提高我们确保食物和血液供应的能力，并且还将进一步了解朊病毒疾病的进展和病理学。

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.