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)使能技术和具体挑战主题06-HG-102：从复杂组织中的单个活细胞获得基因组、蛋白质组和代谢组学数据的技术。也许蛋白质组学领域最大的挑战是开发一种方法，能够报告从复杂的活组织中获得的高时空分辨率的单个细胞或细胞类型中许多不同蛋白质的丰度和翻译后修饰状态。为了迎接这一挑战，需要解决两个根本问题。首先，需要新的和创新的样本收集方法，以便能够快速有效地从嵌入到活组织中的单个细胞中回收材料。其次，需要高度灵敏的分析技术，能够在极小的样本量(1-100个细胞)中以多重方式准确地定量蛋白质。在这里，我们描述了一种名为“显微裂解技术”的新技术，该技术能够从嵌入复杂的活组织中的单个细胞中收集裂解物。这项技术利用裂解缓冲液的流动层流，在大约三到五秒内有效地裂解单个细胞，并以大约两纳升的体积回收裂解物。首先，提出了一种策略，以构建一种使这项技术自动化的仪器。其次，提出了将这项技术与裂解微阵列技术相结合的计划，以便以高度多重和高通量的方式量化蛋白质丰度和翻译后修饰状态。这些开发工作将集中在最具挑战性的组织-哺乳动物大脑，它由数千种不同的细胞类型组成，因此打破了大多数生物化学和蛋白质组学在分子水平上表征它的努力。这里介绍的技术实际上可以在两年的时间框架内开发出来。如果这项工作成功，将在资助期结束时成立一家新公司，将微裂解技术商业化。这项提议的资金将通过立即购买仪器设备、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个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的影响。到目前为止，这些领域的努力要么依赖于生理相关性有问题的培养细胞，要么依赖于由数十到数百种不同类型的细胞组成的整个组织裂解物。微裂解技术将使对几乎任何固体组织中的生物分子进行生理学相关研究成为可能。生物化学中最大的挑战之一是同时使用单个细胞或从复杂的活组织中获得的单一细胞类型来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“显微裂解技术”，它能够自动收集和定量分析嵌入急性脑片中的单个细胞。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固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.