A New Approach For Single-cell Proteomics

Within the decade after the completion of the human genome project,"omics" research, which mainly focuses on nuclear acid, protein, metabolite and their interaction networks, is now becoming an important tool to discover the chemical essence of life and to accelerate the understanding of molecular mechanism of life. In recent years, new phenomena discovered in cancer and stem cell research field sped up the invention and development of new single-cell analytical technologies, such as microfluidics, flow cytometry, mass spectroscopy, Raman spectroscopy and chemical cytometry. These new analytical methods have shown advantages of high sensitivity, throughput and resolution, facilitating single-cell proteomic research.In this thesis, we proposed a novel approach for single-cell proteomics by bridging flow cytometry with chemical cytometry, employing a recently developed activity-based probe (ABP) technique. A capillary electrophoresis and laser induced fluorescence (CE-LIF) detection system was developed with high sensitivity under the principle of single-cell proteomics. Combining with the high specificity and fluorescence of ABP, we were able to perform analysis of low-copied membrane receptor (GABAB receptor) and cysteine cathepsins inside the lysosome at the single-cell level.(1) A CE-LIF system was developed for single-cell analysis. A negative pressurized device facilitated the injection of target single cell into capillary. The inner wall of capillary was coated with a thin layer of polyacrylamide which resisted the adsorption of protein and enhanced the efficiency of separation. Then we verified the separation efficiency by separating protein marker. The concentration detection limit was4×10-12M for fluorescein, which was sufficient for single-cell detection. High repeatability was also achieved both in capillary zone electrophoresis and capillary sieving electrophoresis modes. The optimized sensitivity, separation efficiency and repeatability allowed the developed system to be used for single-cell proteomic research.(2) Based on the CE-LIF system, we developed a ABP based new approach for single-cell proteomic, and performed single-cell functional proteomic redearch on low-copied GABAB receptor families. Three cell lines (HEK, MEF, CHO cells) and primary CGN cells extracted from mouse cerebella were labeled by ABP and delivered to single-cell capillary electrophoresis analysis by negative pressure. We found that single HEK cell expressed proteins with ABP bonding ability, then we tranfected HEK cell with plasmid to overexpress GB1subunit and confirmed the existence of GABAB receptor. We found significant difference among cell lines by coamparing the results of single cells from different cell lines. Also, single cells from the same cell lines and from primary CGN cells exhibited heterogeinity in expression of GABAB receptor. In further single-cell drug competitive bonding experiments carried out upon single CGN cells, the results were consistent with common drug assays performed. The drug competitive bonding experiments provided us with a new method for drug development, drug screening and drug target finding in more precise levels in single cells(3) As mentioned above, a simple co-incubation would be sufficient for ABP labeling of membrane proteins like GABAB receptor. While in the case of enzymes in the cell plasma and deep inside organelles, the cell membrane would be the first blockade to prevent the ABP from entering. Here, we used a cell-permeable ABP molecule to open up the avenue for fluorescence labeling of cysteine cathepsin in lysosome. The basic steps and key parameters for labeling cysteine cathepsin in live cells were firstly determined by real-time fluorescence imaging. Results from single-cell analysis were compared with those from SDS-PAGE based analysis of multiple cells. The bands from SDS-PAGE were analyzed by MS. Results showed that the expression level and activity of cysteine cathepsins in some single cells was similar to those in multiple cells, while other single cells exhibited different activities. It suggested that single-cell analysis is able to reveal extreme phenomena that happened only in minority of cells, leading to new discoveries.Generally, we proposed a new single-cell proteomic approach that was able to investigate low-copied membrane receptor and protease hard to label in cell organelles. Thus provided a new powerful tool for single-cell proteomics research.