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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

基本信息

批准号：
8538932
负责人：
Kermit King Murray
金额：
$ 17.61万
依托单位：
LOUISIANA STATE UNIV A&M COL BATON ROUGE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8538932
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 public health relevance research study single cell analysis small molecule tool

项目摘要

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.

描述（由申请人提供）：生物系统的许多生化分析是在细胞组上进行的，基于这样的假设：细胞足够相似，来自细胞组的整体平均值将产生有用的结果。然而，这种方法可能掩盖单个细胞潜在的异质性，并掩盖基因表达、蛋白质水平和小分子分布的导入和差异。如果要实现单细胞生化分析，就必须将现代分析方法的灵敏度和空间分辨率提高到新的水平。质谱是细胞生物化学的关键分析技术，但在采样规模方面存在技术障碍，必须克服这些障碍才能充分发挥单细胞的潜力。这项研究的目标是构建和测试与电喷雾电离质谱联用的单细胞和组织纳米级激光烧蚀采样系统。该系统使用无孔径近场激光烧蚀将肽、蛋白质和其他生物分子转移到用于超高灵敏度的微滴中电喷雾电离。该项目分为两个部分：(1) 采样系统设置、验证和生物分子标准测试，这将建立检测限和空间分辨率基准；(2) 细胞和组织概念验证测试，这将建立检测和空间分辨率的工作极限，并提供基准样本复杂性确定，以帮助设计后续实验。该项目开发的纳米级激光烧蚀采样不仅可用于质谱分析，还可用于微流体技术。带有液滴捕获的激光烧蚀采样将为微流体装置中的空间分辨采样提供一种新方法。它还将允许在质谱成像中添加分离步骤，从而能够对组织的次要生物分子成分进行成像。