Novel in situ proteomics methods to classify cell types in Alzheimer’s brains

项目摘要 在完整组织的单个细胞中进行全面的原位蛋白质图谱分析有望解开 生物学和医学中的重大谜团，因为它可以揭示空间组织、基因表达 多细胞生物体中不同细胞类型的调节和相互作用，如脑组织，固体 肿瘤和发育中的胚胎。然而，这一梦想在很大程度上仍未实现，因为目前的 蛋白质成像方法受到检测灵敏度低、样品吞吐量和多路复用度的限制 容量。现有方法的这些根本局限性阻碍了它们在福尔马林分析中的应用。 固定石蜡包埋(FFPE)组织，这是最常见的保存类型的临床标本。我们的 首要目标是开发一种高度敏感和高通量的综合蛋白质方法。 FFPE组织的原位图像分析。为实现这一目标，我们提出了以下三个具体目标。目标1： 开发可切割的荧光抗体，用于FFPE的高通量和高度多元化的蛋白质谱分析 纸巾。目的2：建立用于FFPE组织综合蛋白质谱分析的高灵敏度探针。目标3： 单细胞综合蛋白对正常人和阿尔茨海默病患者脑内不同细胞类型的分类 侧写。如果成功，这些基于可切割探针的技术将对理解产生广泛的影响， 诊断和治疗复杂疾病，如神经退行性疾病和癌症。通过映射 单个细胞内的调控网络和组织中细胞之间的相互作用，我们将 加强我们对疾病分子机制的理解，并可能发现治疗疾病的新药物靶点 更有效的细胞靶向治疗。通过准确地指出不同物种丰度和位置的变化 单细胞中的蛋白质，新的生物标记物可以被发现来改变分子诊断和治疗 监控。该项目中开发的方法可以很容易地被其他实验室采用，并将 向公众开放。

Project Summary Comprehensive in situ protein profiling in individual cells of intact tissues holds great promise to unlock major mysteries in biology and medicine, since it can reveal the spatial organization, gene expression regulation, and interactions of the diverse cell types in multicellular organisms, such as brain tissues, solid tumors and developing embryos. However, this dream remains largely unrealized as the current multiplexed protein imaging methods are limited by their low detection sensitivity, sample throughput and multiplexing capacity. These fundamental limitations of the existing methods hinder their applications to profile formalin- fixed paraffin-embedded (FFPE) tissues, which are the most common type of preserved clinical samples. Our overarching goal is to develop a highly sensitive and high-throughput approach for comprehensive protein profiling in FFPE tissues in situ. To achieve this goal, we propose the following three specific aims. Aim 1: develop cleavable fluorescent antibodies for high-throughput and highly multiplexed protein profiling in FFPE tissues. Aim 2: develop highly sensitive probes for comprehensive protein profiling in FFPE tissues. Aim 3: classify the diverse cell types in human normal and Alzheimer’s brains by single-cell comprehensive protein profiling. If successful, these cleavable probes based technologies will have broad impact on understanding, diagnosis and treatment of complex diseases, such as neurodegenerative diseases and cancer. By mapping regulatory networks within individual cells and interrogating interactions among cells in a tissue, we will enhance our understanding of the molecular mechanisms of diseases, and may discover novel drug targets for more effective cellular targeted therapy. By pinpointing alterations in the abundances and positions of various proteins in single cells, new biomarkers can be identified to transform molecular diagnosis and treatment monitoring. The methods developed in the project can be readily adopted by other labs, and will be made publicly available.