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

项目摘要 完整组织的单个细胞中的全面原位蛋白质谱分析为解开 生物学和医学上的重大谜团，因为它可以揭示空间组织，基因表达 调节和多细胞生物体中不同细胞类型的相互作用，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，因为目前的多路复用 蛋白质成像方法受到其低检测灵敏度、样品通量和多路复用的限制 容量现有方法的这些基本局限性阻碍了它们在福尔马林- 固定石蜡包埋（FFPE）组织，其是保存的临床样品的最常见类型。我们 总体目标是开发一种高灵敏度和高通量的方法， 在FFPE组织中原位分析。为了实现这一目标，我们提出以下三个具体目标。目标1： 开发可切割的荧光抗体，用于FFPE中的高通量和高度多重蛋白质分析 组织中目的2：开发高灵敏度的FFPE组织蛋白质谱探针。目标3： 用单细胞综合蛋白对正常人和阿尔茨海默病患者脑内的多种细胞类型进行分类 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 在单个细胞内的调控网络和询问组织中细胞之间的相互作用，我们将 提高我们对疾病分子机制的理解，并可能发现新的药物靶点， 更有效的细胞靶向治疗。通过精确定位不同物种的丰度和位置的变化， 单细胞中的蛋白质，可以识别新的生物标志物，以改变分子诊断和治疗 监测.该项目中开发的方法可以很容易地被其他实验室采用，并将在 公开可用。

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.