Analysis Of C17.2 Neural Stem Cells Surface Proteome And N-linkd Glycoproteome

Neural stem cells are the multipotential,self-renewing cells in many regions of the mammalian central nervous system. They are able to differentiate into neurons, astrocytes and oligodendrocytes under certain conditions. According to their capacity of self-renewal, multipotency and low immunogenic properties as well as migration and integration with the host tissue, they can be used for gene therapy and rehabilitation of a number of central nervous system injury or diseases. Searching for new signal pathway involved in differentiation of neural stem cells and clarifying the mechanism of pluripotency (self-renewal or proliferation) and differentiation is one of the most important areas in the research of neural stem cells. In the process of their self-renewal and differentiation, plasma membrane proteins play pivotal role which transmiss signal transduction as the starting position according to external micro-environmental changes. Lots of studies have shown that various types of ion channels, antigen molecules, and surface receptors of plasma membrane play very important roles in cell proliferation, migration, apoptosis and differentiation. Our experiment aimed at studying the plasma membrane proteins of neural stem cells. By aqueous two-phase partition, we purified plasma membrane of mNSCs(line C17.2), which are immortalized neural stem cell lines which were transfected v-myc gene.Through SDS-PAGE, trypsin digestion, LC-MS/MS and bioinformatics, we identified 222 plasma membrane proteins. Transport proteins accounted for the largest amouts. A great many receptors, signals and enzymes were also found, which contributed to the further study of neural stem cells and application. Many proteins such as Presenilin-1, Nicastrin, Cell adhesion molecule 1 were related to proliferation and differentiation of neural stem cells. We identified and analysed PM proteins of C 17.2 neural stem cells for the first time.The date can provide application value for the research in proliferation and directional differentiation as well as repair of degenerative diseases.Most membrane proteins are glycosylated protein. Cell surface glycoproteins have important biological functions, including mutual recognition between cells, adhesion, signal reception, communications, immune response, etc. A variety of known diseases biomarkers and important molecule antibody molecules are glycoproteins. However, based on the enrichment method of plasma membrane and mass spectrometry technology currently, high-abundance proteins were identified substantially rather than low-abundance glycoproteins. Therefore, efficient glycoprotein or glycopeptides enrichment and removal of high-abundance proteins with non-glycosylation can greatly increase the efficiency of glycoproteins identification.In our study, We applyed the enrichment and identification technology of cell surface glycoprotein(Cell Surface Capturing, CSC-technology) that selectively enriched glycopeptides of C17.2 neural stem cells exposed to the cell exterior, which involved biotinylation of cell surface glycoproteins and affinity enrichment of the membrane glycoproteins. By analyzing only the N-glycosites, the sample complexity was drastically reduced and a relative large abundance range of the cell surface proteins were analyzed by mass spectrometry.188 glycopeptides with 200 glycosylation sites were identified,which resulted in the identification of 99 membrane glycoproteins, including 26 CD molecules.140 new N-glycosites were determined experimentally, including the new N-glycosites of Neural cell adhesion molecule L1,Eph receptor and Neuropilin-1,which have been reported to be involved in proliferation and differentiation of neural stem cells. A great many low-density glycoproteins were identified such as Interleukin-6 receptor and CD276.Therefore, we have built up a C17.2 membrane glycoprotein database for the first time. The cell surface glycoproteins had a great value for analysis of directional differentiation of neural stem cells and neurologic disease. Besides,the data had the potential to facilitate biomarker discovery. Futher more,only 28 glycoproteins were in the data of the plasma membrane proteins using aqueous two-phase partition, which added the data of neural stem cells plasma membrane proteins tremendously.