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

微裂解技术：在活组织中实现细胞类型特异性蛋白质组学

• 批准号: 8119843
• 负责人: GAVIN MACBEATH
• 金额: $ 1.12万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8119843
• 关键词: Microlysis; Technology; Enabling; Cell; Type

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.

描述（由申请人提供）：此申请解决广泛的挑战领域（06）启用技术和特定挑战主题06-HG-102：从复杂组织中各个活细胞中获得基因组，蛋白质组学和代谢组数据的技术。蛋白质组学领域中最大的挑战也许是开发可以报告的方法，以报道以高空间和时间分辨率获得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，以使能够从嵌入活组织中的单个细胞中快速有效地恢复材料。其次，需要高度敏感的分析技术，可以以极小的样本量（1-100个细胞）以多重方式准确地量化蛋白质。在这里，描述了一种新技术 - 称为“小溶解技术”，该技术可以从嵌入复杂的活组织中的单个细胞中收集裂解物。该技术使用移动层流的裂解缓冲液流量在大约三到五秒钟内有效地裂解单个单元，并在大约两个纳米含量的体积中恢复裂解物。首先，提出了一种策略来构建一种自动化这项技术的工具。其次，提出了将这项技术与裂解物微阵列技术相结合的计划，以便以高度多重的高通量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组织的最具挑战性的哺乳动物大脑上，哺乳动物大脑包括成千上万种不同的细胞类型，因此无视生化和蛋白质组学在分子水平上表征它的最大生化和蛋白质组学努力。这里提出的技术可以在两年的时间内实际开发。如果这项工作成功，将在资金期结束时成立一家新公司，以将小溶解技术商业化。该提案的资金将通过立即获得仪器，通过雇用两个博士后研究员以及成立新公司来刺激经济，从而创造其他工作。从科学的角度来看，从嵌入复杂生活组织中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的领域产生深远影响。迄今为止，这些领域的努力依赖于具有可疑生理相关性的培养细胞，或者是整个组织裂解物，其中包括数十种到数百种不同的细胞类型。微溶解技术将使几乎任何固体组织中生物分子的生理相关研究。生物化学中最大的挑战之一是使用单个细胞或从复杂的活组织中获得的单细胞类型同时研究数十个到数百个分子。在这里，我们提出了一种称为“小溶解技术”的新技术，该技术可以对嵌入急性脑切片中的单个细胞进行自动收集和定量分析。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以生理相关的方式研究生物分子几乎任何固体组织。