Equipment Supplement Request for a Research Resource for Ultra-sensitive High Throughput Proteomics

超灵敏高通量蛋白质组学研究资源的设备补充请求

• 批准号: 10398678
• 负责人: RICHARD D SMITH
• 金额: $ 23.3万
• 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/10398678
• 关键词: Address; Administrative Supplement; Automobile Driving; Biological; Biomedical Research; Cell Separation; Cells; Clinical; Collaborations; Complement; Coupled; Detection; Development; Devices; Equipment; Fluorescence-Activated Cell Sorting; Fractionation; Gases; Goals; Ions; Letters; Life; Liquid Chromatography; Mammalian Cell; Mass Spectrum Analysis; Measurement; Methods; Microfluidics; Phase; Phosphorylation; Post-Translational Protein Processing; Process; Proteins; Proteome; Proteomics; Resolution; Resources; Retinal blind spot; Robotics; Sample Size; Sampling; Speed; Structure; Surface; System; Technology; Time; Tissues; base; cell type; commercialization; improved; ion mobility; ionization; laser capture microdissection; magnetic beads; mass spectrometer; nano-electrospray; nanoDroplet; novel; parent grant; ultra high resolution

Abstract Parent grant 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.

摘要