质谱单细胞分析方法的建立及其应用

发布时间：2018-03-27 15:42

本文选题：单细胞　切入点：代谢物　出处：《清华大学》2015年博士论文

【摘要】：由于不同的细胞个体之间具有差异性,为了如实地反映细胞在结构和功能上对生物系统的正常运转所起到的作用,就必须从单细胞水平上对细胞中物质的组成和含量进行分析研究,因而单细胞分析的概念在近年来广受关注。但是单细胞分析研究却由于细胞的极小体积,细胞中极多的物质种类和极少的物质含量而成为具有挑战性的课题。本文针对单细胞分析的难点,设计了两种质谱单细胞分析方法,并将这两种方法应用到实际的细胞代谢物检测中。主要研究成果如下:1.提出了溶剂辅助电场诱导解吸附/离子化(SAEFIDI)质谱分析方法,实现了小体积高盐样品中痕量待测分子的检测。该方法利用了盐和待测分子在结晶时自发分离的现象,在Nano-tip中实现了pL级样品中盐和待测分子的分离;并使用SAEFIDI对Nano-tip顶端的待测分子进行原位解吸附/离子化,避免了对待测分子的稀释,实现了高灵敏度的检测。此外,由于分离过程是自发发生的,有效地避免了人为操作可能带来的污染和样品损失,进一步保证了检测灵敏度。2.将所建立的SAEFIDI质谱分析方法应用于动物细胞分裂过程中重要代谢物的含量变化研究中。在三维平台的帮助下,用Nano-tip将特定体积的溶剂滴加到细胞表面,对细胞中的小分子代谢物进行萃取。之后将萃取得到的样品溶液吸回到Nano-tip中,并利用SAEFIDI实现样品的检测。研究发现,在细胞分裂过程中,多种代谢物的含量均有显著变化。此外,我们还通过同位素标记法对细胞中三磷酸腺苷(ATP)、二磷酸腺苷(ADP)和单磷酸腺苷(AMP)的合成代谢进行了研究。结果显示ATP、ADP和AMP的生物合成与葡萄糖的磷酸戊糖途径(PPP)密切相关。3.建立了基于探针电喷雾离子化(PESI)的质谱单细胞分析方法,并在单细胞水平和亚细胞水平上对洋葱细胞中的代谢物进行了检测。该方法使用尖端尺寸仅有1~5μm的金属钨针作为富集探针,在三维移动平台的精确操控下,刺入细胞中的特定部位富集代谢物。然后通过PESI对探针上所富集的代谢物进行原位解吸附/离子化,并与质谱联用进行检测。结果显示,洋葱鳞茎中不同种类的细胞在物质的组成和含量上均有显著差异。而同种类的细胞虽然具有相近的物质组成,但是在物质的含量仍有一定差别。亚细胞水平的检测结果则显示,在洋葱细胞的细胞核与细胞质中,物质的组成和含量均有差异。
[Abstract]:Because of the difference between different cell individuals, in order to reflect the function of cell in structure and function to the normal operation of biological system, It is necessary to analyze the composition and content of substances in cells at the single cell level, so the concept of single cell analysis has attracted much attention in recent years. However, the study of single cell analysis is due to the very small volume of cells. In this paper, two methods of mass spectrometry single cell analysis are designed for the difficulty of single cell analysis. These two methods have been applied to the detection of cell metabolites. The main research results are as follows: 1. A solvent-assisted electric field induced desorption / ionization SAEFIDI mass spectrometry method is proposed. The detection of trace molecules in small volume and high salt samples was carried out. The method utilized the phenomenon of spontaneous separation of salt and the molecules to be tested during crystallization, and achieved the separation of salt from the molecules to be tested in Nano-tip. SAEFIDI is used for in situ desorption / ionization of the molecules to be tested at the top of Nano-tip, which avoids dilution of the tested molecules and achieves high sensitivity detection. In addition, the separation process occurs spontaneously. Effectively avoiding possible contamination and sample loss caused by human operations, The SAEFIDI mass spectrometry method was applied to study the changes of important metabolites in the course of animal cell division. With the help of three-dimensional platform, A specific volume of solvent was added to the cell surface with Nano-tip to extract the metabolites of small molecules in the cell. Then the extracted sample solution was absorbed back into the Nano-tip, and the sample was detected by SAEFIDI. During cell division, the contents of various metabolites changed significantly. In addition, The biosynthesis and metabolism of adenosine triphosphate ATP (adenosine diphosphate) and adenosine monophosphate (AMP) in cells were also studied by isotope labeling. The results showed that the biosynthesis of ATP ADP and AMP was closely related to the glucose pentose phosphate pathway. A single cell mass spectrometric analysis method based on probe electrospray ionization (PESI) was established. The metabolites in onion cells were detected at the single cell level and subcellular level. The method used a metal tungsten needle with a tip size of only 1 ~ 5 渭 m as a enrichment probe under the precise manipulation of a three-dimensional mobile platform. In situ desorption / ionization of the metabolites enriched on the probe by PESI, and detection by mass spectrometry. There were significant differences in the composition and content of substances between different kinds of cells in onion bulb, while the same type of cells had similar material composition, but there were still some differences in the content of substance. The results of subcellular level showed that, The composition and content of substances in the nucleus and cytoplasm of onion cells are different.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q2-33;O657.63

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