Development Of Mass Spectrometric Single Cell Analysis Methods And Their Applications

Biological systems are composed of cells with heterogeneity. To better understand the structural and functional roles that cells play in biological systems, we need to analyze them on an individual basis qualitatively and quantitatively. The concept of single cell analysis has attracted extensive attentions in recent years. It is believed to be beneficial to a variety of potential applications. However, the research of single cell analysis is challenging due to the very small volume of cells, the large numbers of metabolites in cells and the so little amount of materials that could be obtained from an individual cell for detection. To meet this challenge, here, we developed two mass spectrometric single cell analysis methods for the detection of metabolites in single cells. The main contents of the present work are listed as follows:1. A novel ionization method was developed, namely solvent assisted electric field induced desorption/ionization(SAEFIDI). Successful detection of trace analytes in picoliter samples with high concentrations of salts was achieved using this method. It was based on the spontaneous separation of biomolecules from salts during crystallization of the salts. As the desalting process was accomplished spontaneously within the tip of a quartz pipette, potential sample loss, dilution and contaminants were avoided. Subsequent in situ desorption/ionization of the separated biomolecules was achieved using SAEFIDI, which avoided dilution of the sample and ensured sensitive detection of biomolecules.2. We made use of SAEFIDI and investigated the content changes of some important metabolites in mammalian cells during division. A Nano-tip was used to drop a certain volume of solvent onto the surface of the targeted cell to extract metabolites from the cell. The Nano-tip was precisely controlled by a three-dimensional manipulator. After extraction, the obtained solution was sucked back into the Nano-tip. Subsequent in situ desorption/ionization of the sample from the Nano-tip was achieved by SAEFIDI coupled with mass spectrometry. According to the results, a variety of metabolites were found to have significant changes during cell division. Furthermore, isotopic labeling was used to investigate the synthetic pathways of adenosine triphosphate(ATP), adenosine diphosphate(ADP) and adenosine monophosphate(AMP). We found that the synthesis of ATP, ADP and AMP was closely related to the pentose phosphate pathway(PPP).3. A probe electrospray ionization(PESI) based single cell analysis method was developed. It was further used for the detection of metabolites in onion cells at cellular and sub-cellular level. A tungsten probe with a tip diameter of 1~5 μm was used. It was controlled by a three-dimensional manipulator to precisely insert into the targeted cell for the enrichment of metabolites. Afterwards, the enriched metabolites were directly desorbed/ionized in situ from the probe by PESI for mass spectrometry(MS) detection. According to the results, significant chemical diversity was observed between different kinds of cells. For cells of the same kind, the chemical composition was similar. However, the relative abundance of metabolites still differed from cell to cell. At last, the chemical composition of sub-cellular zones, including nucleus and cytoplasm, was investigated. Obvious chemical diversity was observed.