Nanoscale Laser Ablation Capture Mass Spectrometry for Single Cell Proteomics

描述（由申请人提供）：基于细胞足够相似的假设，对细胞组进行生物系统的许多生化分析，从而细胞组的总体平均值将产生有用的结果。然而，这种方法可能会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输入和差异。如果要实现单细胞生化分析，就必须将现代分析方法提高到新的灵敏度和空间分辨率水平。质谱是细胞生物化学的关键分析技术，但要充分利用单细胞，必须克服采样规模方面的技术障碍。 本研究的目的是建立和测试一个系统或纳米尺度的单细胞和组织的激光消融采样耦合电喷雾电离质谱。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品测试，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限，并提供基准样品复杂性测定，这将有助于设计后续实验。 在这个项目中开发的纳米级激光烧蚀取样不仅将在质谱分析中应用，而且将在微流体中应用。激光烧蚀采样与液滴捕获将提供一种新的方法，空间分辨采样到微流控装置。它还将允许将分离步骤添加到质谱成像中，这将使得能够对组织的次要生物分子组分进行成像。

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.