Microlysis Technology: Enabling Cell Type-Specific Proteomics in Living Tissue

描述（由申请人提供）：本申请涉及广泛的挑战领域（06）使能技术和特定挑战主题06-HG-102：从复杂组织中的单个活细胞获得基因组、蛋白质组和代谢组数据的技术。也许在蛋白质组学领域的最大挑战是开发的方法，可以报告的丰度和翻译后修饰状态的许多不同的蛋白质在一个单一的细胞或细胞类型获得高空间和时间分辨率从复杂的，活的组织。为了应对这一挑战，需要解决两个基本问题。首先，需要新的和创新的样品收集方法，以实现从嵌入活组织中的单细胞中快速有效地回收材料。其次，需要高灵敏度的分析技术，可以在极小的样品尺寸（1-100个细胞）中以多重方式准确定量蛋白质。在这里，描述了一种新技术-称为“微裂解技术”-能够从嵌入复杂活组织的单细胞中收集裂解物。该技术使用裂解缓冲液的移动的层流，以在大约3至5秒内有效地裂解单个细胞，并以大约2纳升的体积回收裂解物。首先，提出了一种策略，以建立一个仪器，自动化这项技术。其次，提出了一个计划，以耦合这种技术与裂解物微阵列技术，以量化蛋白质丰度和翻译后修饰状态的高度复用和高通量的方式。这些开发工作将集中在最具挑战性的组织，即哺乳动物大脑，它由数千种不同的细胞类型组成，因此无法在分子水平上对其进行表征。这里介绍的技术实际上可以在两年的时间内开发出来。如果这项工作取得成功，将在资助期结束时成立一家新公司，将微裂解技术商业化。对这一提案的资助将通过立即购买仪器、雇用两名博士后研究员和成立一家新公司来刺激经济，从而创造更多的就业机会。在科学层面上，从嵌入复杂活组织的单细胞中收集裂解物的能力将对基因组学、蛋白质组学和代谢组学领域产生深远影响。迄今为止，在这些领域的努力依赖于培养的细胞，其具有可疑的生理相关性，或者依赖于全组织裂解物，其包含数十至数百种不同的细胞类型。微裂解技术将使几乎任何实体组织中的生物分子的生理学相关研究成为可能。生物化学中最大的挑战之一是使用从复杂的活组织中获得的单细胞或单细胞类型同时研究数十到数百个分子。在这里，我们提出了一种新的技术，称为“微裂解技术”，它使嵌入在急性脑切片的单细胞的自动化收集和定量分析。这项技术将对基因组学，蛋白质组学和代谢组学领域产生深远的影响，因为它将使研究人员能够在几乎任何实体组织中以生理相关的方式研究生物分子。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies and Specific Challenge Topic 06-HG-102: Technologies for obtaining genomic, proteomic, and metabolomic data from individual viable cells in complex tissues. Perhaps the greatest challenge in the area of proteomics is to develop methods that can report on the abundances and post-translational modification states of many different proteins in a single cell or cell type obtained with high spatial and temporal resolution from complex, living tissue. There are two fundamental issues that need to be addressed in order to meet this challenge. First, new and innovative sample collection methods are needed to enable the fast and efficient recovery of material from single cells embedded in live tissue. Second, highly sensitive analytical techniques are needed that can accurately quantify proteins in a multiplexed fashion in extremely small sample sizes (1-100 cells). Here, a new technology - termed "microlysis technology" - is described that enables the collection of lysates from single cells embedded in complex, living tissue. This technology uses mobile laminar flow of lysis buffer to efficiently lyse a single cell in approximately three to five seconds and to recover the lysate in a volume of approximately two nanoliters. First, a strategy is presented to build an instrument that automates this technology. Second, a plan is presented to couple this technology with lysate microarray technology in order to quantify protein abundances and post-translational modification states in a highly multiplexed and high-throughput fashion. These development efforts will be focused on the most challenging of tissues, the mammalian brain, which comprises thousands of distinct cell types and hence has defied most biochemical and proteomics efforts to characterize it at the molecular level. The technology presented here can realistically be developed in a two-year timeframe. If this work is successful, a new company will be launched at the end of the funding period to commercialize microlysis technology. Funding of this proposal will stimulate the economy through the immediate acquisition of instrumentation, through the hiring of two postdoctoral fellows, and through the founding of a new company, thereby creating additional jobs. On a scientific level, the ability to collect lysates from single cells embedded in complex living tissue will have a profound effect on the fields of genomics, proteomics, and metabolomics. To date, efforts in these areas have relied either on cultured cells, which have questionable physiological relevance, or on whole tissue lysates, which comprise dozens to hundreds of distinct cell types. Microlysis technology will enable the physiologically relevant study of biomolecules in virtually any solid tissue. One of the greatest challenges in biochemistry is to study dozens to hundreds of molecules simultaneously using single cells or in single cell types obtained from complex, living tissue. Here, we propose a new technology, termed "microlysis technology", which enables the automated collection and quantitative analysis of single cells embedded in acute brain slices. This technology will have a profound impact on the fields of genomics, proteomics, and metabolomics since it will enable researchers to study biomolecules in a physiologically relevant fashion in virtually any solid tissue.