Nanoscale Laser Ablation Capture Mass Spectrometry for Single Cell Proteomics

用于单细胞蛋白质组学的纳米级激光烧蚀捕获质谱法

• 批准号: 8413991
• 负责人: Kermit King Murray
• 金额: $ 16.93万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8413991
• 关键词: Ablation; Benchmarking; Biochemical; Biochemistry; Biological; Blood capillaries; Caliber; Cells; Cellular Structures; Chemistry; Coupled; Cryoultramicrotomy; Deposition; Detection; Development; Electrospray Ionization; Film; Gene Expression; Goals; Health; Heterogeneity; Human; Image; Individual; Ions; Lasers; Masks; Mass Spectrum Analysis; Measurement; Methods; Microfluidic Microchips; Microfluidics; Minor; Mus; Needles; Optics; Outcome; Particle Size; Peptides; Performance; Photography; Physiologic pulse; Pituitary Gland; Positioning Attribute; Process; Property; Proteins; Proteomics; Radial; Research; Resolution; Sampling; Scanning Probe Microscopes; Silver; Slide; Solvents; Source; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staging; System; Techniques; Testing; Tissues; Validation; Work; analytical tool; base; biological systems; brain tissue; capillary; cellular imaging; design; experience; ion source; ionization; mass spectrometer; nano-electrospray; nanoscale; nanosecond; prevent; research study; single cell analysis; small molecule; tool

DESCRIPTION (provided by applicant): Many biochemical analyses of biological systems are performed on groups of cells based on the assumption that the cells are sufficiently similar that an ensemble average from the cell group will yield a useful result. However, this approach can obscure the underlying heterogeneity of the individual cells and masks import and differences in gene expression, protein levels, and small-molecule distributions. Bringing modern methods of analysis to new levels of sensitivity and spatial resolution is necessary if single cell biochemica analysis is to be achieved. Mass spectrometry is a key analysis technique for cell biochemistry, but there are technological barriers in sampling scale that must be overcome for it to be used to its full potential with single cells. The goal of this research is to construct and test a system or nanometer scale laser ablation sampling of single cells and tissue coupled with electrospray ionization mass spectrometry. This system uses apertureless near-field laser ablation to transfer peptides, proteins and other biomolecules to a microdroplet that is used for ultra high sensitivity electrospray ionization. The project is divided into two components (1) sampling system setup, validation, and testing with biomolecule standards, which will establish detection limit and spatia resolution benchmarks, and (2) proof of concept testing with cells and tissue, which will establish a working limit of detection and spatial resolution as well as provide a benchmark sample complexity determination that will aid in designing follow-on experiments. Nanoscale laser ablation sampling developed in this project will have applications not only in mass spectrometry but also in microfluidics. Laser ablation sampling with droplet capture will provide a new method for spatially resolved sampling into a microfluidic device. It will also allow a separation step to be added to mass spectrometry imaging that will enable the imaging of minor biomolecule components of tissue. PUBLIC HEALTH RELEVANCE: Single cell analysis is necessary because the ensemble average of cell mixtures obscures important information about human health therefore it is necessary to develop new single cell analysis techniques. Mass spectrometry is one of the most important analysis tools but technological barriers prevent sampling and imaging at the single cell level if large biomolecules such as peptides and proteins are the target analyte. The goal of this research is to develop nanometer scale sampling for mass spectrometry using near-field optics and laser ablation.

描述（由申请人提供）：基于这样的假设，即细胞足够相似，即细胞组的平均平均值将产生有用的结果，对生物系统进行了许多生化分析。但是，这种方法可以掩盖单个细胞的潜在异质性，并掩盖基因表达，蛋白质水平和小分子分布的进口和差异。如果要实现单细胞生物化学分析，则必须将现代分析方法提高到新的灵敏度和空间分辨率。质谱法是细胞生物化学的一项关键分析技术，但是在采样量表中存在技术障碍，必须克服它才能将其全部用于单个细胞的潜力。 这项研究的目的是构建和测试单个细胞和组织与电喷雾电离质谱的单细胞和组织的系统或纳米尺度激光消融采样。该系统使用无孔的近场激光消融来将肽，蛋白质和其他生物分子转移到用于超高灵敏度的微颗粒上 电喷雾电离。该项目分为两个组件（1）采样系统的设置，验证和使用生物分子标准标准进行测试，这些标准将建立检测极限和空间分辨率基准，以及（2）用细胞和组织进行概念验证证明，这将建立检测和空间分辨率的工作限制，并提供基本的复杂性确定，并提供了辅助效果。 在该项目中开发的纳米级激光消融采样不仅在质谱中，而且还具有微流体的应用。用液滴捕获的激光消融采样将为微流体设备提供一种新的方法，用于空间分辨采样。它还将允许将分离步骤添加到质谱成像中，以实现组织的少量生物分子成分的成像。 公共卫生相关性：单细胞分析是必要的，因为细胞混合物的整体平均值掩盖了有关人类健康的重要信息，因此有必要开发新的单细胞分析技术。质谱是最重要的分析工具之一，但是如果大型生物分子（例如肽和蛋白质）是靶分析物，则技术障碍可以防止单细胞水平采样和成像。这项研究的目的是使用近场光学和激光消融开发质谱法进行纳米尺度采样。