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）：许多生物系统的生化分析是在细胞群上进行的，基于细胞足够相似的假设，从细胞群中获得的总体平均值将产生有用的结果。然而，这种方法可能会模糊单个细胞的潜在异质性，掩盖基因表达、蛋白质水平和小分子分布的输入和差异。如果要实现单细胞生化分析，将现代分析方法提高到新的灵敏度和空间分辨率水平是必要的。质谱法是细胞生物化学的一项关键分析技术，但要充分发挥其在单细胞分析中的潜力，必须克服采样尺度上的技术障碍。本研究的目的是建立和测试一个纳米尺度的单细胞和组织激光烧蚀采样系统，并结合电喷雾电离质谱法。该系统使用无孔近场激光烧蚀将肽、蛋白质和其他生物分子转移到微滴中，用于超高灵敏度