Automated processing and manipulation of small samples for high throughput and ultrasensitive functional proteomics measurements

自动处理和操作小样品，以实现高通量和超灵敏的功能蛋白质组学测量

• 批准号: 10220049
• 负责人: Ryan T Kelly
• 金额: $ 25.11万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-15 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10220049
• 关键词: Address; Automation; Automobile Driving; Biological; Cell Separation; Cells; Chemicals; Clinical; Collaborations; Communities; Complement; Coupled; Coupling; Development; Devices; Evaluation; Fluorescence-Activated Cell Sorting; Fractionation; Gases; Goals; Ions; Isotope Labeling; Liquid Chromatography; Mammalian Cell; Mass Spectrum Analysis; Measurement; Methodology; Methods; Microfluidics; Peptides; Phase; Phosphorylation; Population; Post-Translational Protein Processing; Preparation; Process; Proteins; Proteome; Proteomics; Resolution; Resources; Retinal blind spot; Robotics; Sample Size; Sampling; Structure; Surface; Technology; Time; Tissues; Variant; base; cell type; flexibility; improved; ion mobility; ionization; laser capture microdissection; magnetic beads; miniaturize; nano-electrospray; nanoDroplet; nanoscale

Project Summary – TR&D 1 The overarching goal of the Resource is to dramatically improve on the existing proteomics paradigm by achieving orders-of-magnitude gains in both measurement sensitivity and throughput, as well as to address important measurement `blind spots' and shortcomings of current methods. At present, a minimum of thousands of cells are generally required for in-depth coverage of proteins in a biological sample, precluding many important applications involving extremely small samples, rare cell types or spatially resolved measurements. We have recently developed a Nanodroplet Processing in One-pot for Trace Samples (nanoPOTS) technology which, when coupled with ultrasensitive MS-based measurements, enables effective analysis of as few as 10 mammalian cells. The technology efforts of TR&D 1 will extend this robotic/microfluidic nanoPOTS platform to provide the `up-front' processing required to efficiently handle ultra-small samples (extending to single cells) and deliver these samples optimally to the Structures for Lossless Ion Manipulations (SLIM)-based ion mobility-mass spectrometry (IM-MS) platforms to be further advanced under TR&D 2. The robotic platform will enable samples to be transferred using nanoelectrospray ionization to the SLIM IM-MS platforms with high ionization and utilization efficiencies. The nanoPOTS platform and workflow will be optimized to enable broad proteome coverage for sample sizes in the range of 1-1000 cells so as to enable broad proteome coverage. The efforts will also establish seamless, automated integration of nanoPOTS with the widely used cell isolation technologies of fluorescence activated cell sorting (FACS) and laser-capture microdissection (LCM), enabling ultra-rare cell analysis and high-resolution proteome mapping of clinical tissues. We will also extend nanoPOTS processing to address the inherently small sample sizes and sensitivity challenges associated with functional proteomics measurements, including activity-based proteomics and measurements of key post-translational modifications such as phosphorylation, using a combination of direct surface functionalization, magnetic bead-based workflows within the nanowells and microcolumn fractionation to enrich and process functional protein subpopulations. We aim to reduce required sample sizes for such functional measurements by more than 100-fold. The efficient processing and greatly reduced sample losses enabled by the nanoPOTS platform will complement the large gains in throughput and sensitivity afforded by TR&D 2 and its fast and high-resolution gas-phase separations and manipulations in SLIM. The workflows to be developed will enable both online and offline separations (e.g., liquid chromatography) with SLIM IM-MS using nanoPOTS-prepared samples. The development of this platform will occur in close collaboration with the TR&D 2 and 3 efforts and is key to most of the driving biological projects.

项目概要- TR&D 1 该资源的总体目标是通过以下方式大大改善现有的蛋白质组学范式： 在测量灵敏度和吞吐量方面都实现了数量级的增益， 重要的测量“盲点”和现行方法的缺点。目前， 通常需要数千个细胞来深入覆盖生物样品中的蛋白质， 许多重要的应用涉及极小的样品、罕见的细胞类型或空间分辨 测量.我们最近开发了一种用于痕量样品的一锅法纳米液滴处理 （nanoPOTS）技术，当与基于MS的超灵敏测量相结合时， 分析少至10个哺乳动物细胞。TR&D 1的技术努力将扩展这一点 机器人/微流体nanoPOTS平台，以提供有效处理所需的“前期”处理 超小样品（扩展到单个细胞），并将这些样品最佳地输送到结构， 基于无损离子操作（SLIM）的离子迁移率-质谱（IM-MS）平台将进一步 在TR&D 2下进行。机器人平台将使样品能够使用纳米电喷雾转移 SLIM IM-MS平台具有高电离和利用效率。纳米锅 平台和工作流程将进行优化，以实现广泛的蛋白质组覆盖范围内的样本量 1-1000个细胞，以实现广泛的蛋白质组覆盖。这些努力还将建立无缝的、自动化的 纳米POTS与广泛使用的荧光激活细胞分选的细胞分离技术的集成 流式细胞仪（FACS）和激光捕获显微切割（LCM），使超稀有细胞分析和高分辨率 临床组织的蛋白质组作图。我们还将扩展nanoPOTS处理以解决固有的小型问题 与功能蛋白质组学测量相关的样本量和灵敏度挑战，包括 基于活性的蛋白质组学和关键的翻译后修饰如磷酸化的测量， 使用直接表面功能化、微池内基于磁珠的工作流程的组合 和微柱分级分离以富集和处理功能性蛋白质亚群。我们的目标是减少 这种功能测量所需的样本量增加了100倍以上。高效的处理和 通过nanoPOTS平台实现的大幅降低的样本损耗将补充 TR&D 2及其快速、高分辨率气相分离提供的通量和灵敏度， 在SLIM中操作。待开发的工作流程将实现在线和离线分离（例如， 液相色谱法）与SLIM IM-MS使用纳米POTS制备的样品。发展这个 该平台将与TR&D 2和3的工作密切合作，并且是大多数驱动的关键 生物项目。