Highly sensitive proteomics method to probe cell heterogeneity at single cell resolution

项目摘要/摘要： 随着单细胞转录组分析变得更加容易，单个细胞 现在可以单独分类，使我们能够理解细胞异质性是 神经元发育和肿瘤进化的关键驱动力。然而，出现了 MRNA并不总是伴随着蛋白质的表达，单细胞蛋白质组学将提供更多 精确查看细胞组件和信号，包括关键的翻译后修饰 如磷酸化等事件。 在这里，我们建议结合几种不同的策略来开发高度敏感的 蛋白质组学方法在单细胞分辨率下探测细胞异质性。具体地说，我们将1) 开发化学标记探针以增强多肽的电离；2)开发一种异构体 同位素标记法定量检测不同细胞系中的单个哺乳动物细胞；3) 开发信息学工具和统计模型以解决单细胞的细胞异质性 与非单细胞蛋白质组学数据的分辨率。仅这些建议的方法中的每一种都可以 有可能使我们能够测量单个哺乳动物细胞的蛋白质组。当组合在一起时， 他们将产生一个高度敏感的平台，可能允许我们探测 单个线粒体或细菌水平上的蛋白质组。 这个项目的长期目标是阐明 细胞异质性的起源和意义。这项拟议的研究与公众高度相关 在肿瘤进化、癌症转移、发育神经生物学和 神经退行性疾病。了解细胞异质性将有助于我们开发更多 治疗多种疾病的有效药物。因此，拟议的研究与以下方面有关 NIH使命的一部分，就是促进“基本的创造性发现、创新性研究 战略及其应用，作为最终保护和改善健康的基础“和 这“支持人类疾病的病因、诊断、预防和治疗方面的研究”。 拟议的研究与NIGMS 2015-2020年战略计划有关：“目标1-1： 投资并维持广泛和多样化的高度有价值的研究组合。 1-2：促进调查人员追求新的研究方向、新颖的科学的能力 见解和创新想法“，以及”目标3-1：支持获得基本研究 资源和新技术的开发，使新的科学进步成为可能。

Project Summary/Abstract: As the single-cell transcriptomics analysis becomes more accessible, individual cells can now be classified individually, allowing us to understand cell heterogeneity as one of the key driving forces in neuron development and tumor evolution. However, the presence of mRNA is not always coupled with protein expression, single-cell proteomics will provide a more precise view of the cell components and signaling, including key post-translational modification events such as phosphorylation. Here we propose to combine several different strategies to develop a highly sensitive proteomics method to probe cell heterogeneity at single cell resolution. Specifically, we will 1) develop chemical labeling probes to enhance peptide ionization; 2) develop an isobaric isotopologue labeling approach to quantify single mammalian cell from various cell lines; 3) develop informatics tools and statistical models to resolve cell heterogeneity at single-cell resolution with non-single-cell proteomics data. Each of these proposed methods alone, could potentially enable us to measure the proteome of a single mammalian cell. When combined, they will result in a highly sensitive platform that could potentially allow us to probe the proteome at the level of a single mitochondria or bacteria. The long-term goal of this project is to elucidate the fundamental mechanisms of the origin and implications of cell heterogeneity. This proposed research is highly relevant to public health in the fields of tumor evolution, cancer metastasis, developmental neurobiology and neurodegenerative disease. Understanding cell heterogeneity will help us to develop more effective drug treatments to many diseases. Therefore, the proposed research is relevant to the part of NIH's mission that fosters “fundamental creative discoveries, innovative research strategies, and their applications as a basis for ultimately protecting and improving health” and that “supports research in the causes, diagnosis, prevention, and cure of human diseases.” The proposed research is relevant to the NIGMS 2015-2020 strategic plan: “Objective 1-1: Invest in and sustain a broad and diverse portfolio of highly meritorious research.”, “Objective 1-2: Promote the ability of investigators to pursue new research directions, novel scientific insights and innovative ideas”, and “Objective 3-1: Support access to essential research resources and the development of new technologies that enable novel scientific advances.”