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

项目总结--研发1 该资源的首要目标是通过以下方式显著改进现有的蛋白质组学范式 在测量灵敏度和吞吐量方面实现数量级的增长，以及解决 重要的测量“盲点”和当前方法的缺点。目前，至少有 为了深入覆盖生物样本中的蛋白质，通常需要数千个细胞，从而排除 涉及极小样本、稀有细胞类型或空间分辨的许多重要应用 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 (NanPOTS)技术，当与超灵敏的基于MS的测量相结合时，能够有效地 只对10个哺乳动物细胞进行分析。研发一号的技术努力将延长这一点 机器人/微流控纳米POTS平台，可提供高效处理所需的“前期”处理 超小样品(延伸到单个细胞)，并将这些样品最佳地输送到结构中 基于无损离子操纵(SLIM)的离子迁移率-质谱仪(IM-MS)平台将进一步 研发下的先进2.机器人平台将能够使用纳米电喷雾转移样品 电离到超薄的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，以实现样本大小范围内的广泛蛋白质组覆盖 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、自动化的 纳米POTS与广泛使用的荧光激活细胞分选分离技术的集成 (FACS)和激光捕获显微切割(LCM)，实现超稀有细胞分析和高分辨率 临床组织的蛋白质组图谱。我们还将扩展NanPOTS处理，以解决本质上很小的 与功能蛋白质组学测量相关的样本大小和敏感性挑战，包括 基于活性的蛋白质组学和关键翻译后修饰的测量，如磷酸化， 使用直接表面功能化的组合，基于磁珠的工作流在纳米孔内 和微柱分离，以丰富和处理功能蛋白亚群。我们的目标是减少 这种功能测量所需的样本量增加了100多倍。高效的处理和 由NanPOTS平台实现的大幅降低的样品损失将补充 由RT&D 2及其快速和高分辨率气相分离和 在斯利姆的操纵。要开发的工作流将允许在线和离线分离(例如， 使用纳米POTS制备的样品与超薄IM-MS联用)。这方面的发展 平台将与研发2和3的工作密切合作，是推动大部分工作的关键 生物项目。

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.