Single molecule protein sequencing of alpha-synuclein species for Parkinson's disease diagnostics

帕金森病（Parkinson's disease，PD）是一种神经退行性疾病， 当60%的大脑区域的多巴胺能神经元已经退化时，诊断出来。一 这种难以捉摸的分子生物标志物的有力竞争者是一种神经元蛋白-- α-突触核蛋白， 后修饰的磷酸化同种型（pSer-129）。沿着与其他C端子 这种改性已被发现为不溶性的聚集和纤维状形式， 已故帕金森病人的脑组织疾病的进展和严重程度 也与患者脑脊液（CSF）中的相对水平相关。 然而，目前的技术，如免疫测定和质谱分析， 所需的灵敏度和能力，以准确地定量磷酸化和 同时在临床上有限的样品中，如 脑脊液这种技术限制阻碍了一种强大的 这些涉及的神经元生物标志物的临床测定和验证。 荧光测序是一种新的单分子蛋白质测序技术， 在一个实施例中，本发明提供了一种用于鉴定和定量复杂混合物中的单个蛋白质的并行化平台。的 单分子平台的重要和显著特征是其固有能力， 绝对定量和区分不同种类的肽，包括 磷酸化残基的位置和占有率不同。 透过资助计划第一及第二期，我们建议利用这个平台技术， 用于测量不同种类的α-突触核蛋白的丰度的临床测定 非常有限的生物样本，并将测量结果与患者结果相关联。 在第一阶段，我们将在不同的浓度下加入修饰和未修饰的α-突触核蛋白种类， 按比例加入CSF中，确定试验重现性和重复性（目的1）并获得限度 不同α-突触核蛋白磷酸化种类之间的检测和区分（目的 2）。完成这些目标将建立一个优化的工作流程， 测量患者样本水平所需的样本要求。成功 这两个阶段的完成将导致走向实验室的商业化道路 开发测试或诊断分析。

ABSTRACT Parkinson’s disease (PD) is a neurodegenerative disorder that is typically diagnosed when 60% of a brain region’s dopaminergic neurons have already degenerated. A strong contender for the elusive molecular biomarker is a neuronal protein - α-synuclein and its post-translationally modified phosphorylated isoform (pSer-129). Along with other C-terminal modifications, this modification has been found as the insoluble aggregated and fibrillar form in the brain tissue of deceased Parkinson’s patients. The progression and disease severity has been correlated to its relative levels in patient’s cerebrospinal fluids (CSF) as well. However, current technologies, such as immuno-assays and mass-spectrometry lack the necessary sensitivity and capability to accurately quantify both the phosphorylated and unmodified form of the protein simultaneously and in clinically limited samples, such as the cerebrospinal fluid. This technological limitation has hampered the development of a robust clinical assay and validation of these implicated neuronal biomarkers. Fluorosequencing, a new single molecule protein sequencing technology, is a massively parallelized platform for identifying and quantifying individual proteins in a complex mixture. The important and distinguishing feature of the single molecule platform is its inherent ability to absolutely quantify and discriminate the different species of peptides, including the species differing in the positions and the occupancy of phosphorylated residues. Through the grant phase I and II, we propose to utilize this platform technology to develop a clinical assay for measuring the abundances of the different species of α-synuclein from extremely limited biological samples and correlating the measurements with patient outcomes. In phase I, we will spike-in modified and unmodified alpha-synuclein species at different proportions into CSF, establishing assay reproducibility and replicability (aim 1) and obtain limits of detection and discrimination between the different α-synuclein phosphorylated species (aim 2). Completing these objectives will establish an optimized work-flow and the determination of the sample requirements necessary for measuring the levels in patient samples. Successful completion of the two phases would result in a commercialization path towards a laboratory developed test or diagnostic assay.