Identification of the specific signaling mediated by kinase and protease crosstalks in glioma stem cells using a unique integration method of phosphoproteomics and N-terminomics

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The six GSC clones established from GBM cell lines were used for profiling their global proteome by MS-based proteomics. We firstly examined the identified number of proteins in urea or phase transfer surfactant-aided (PTS) trypsin digestion and concluded that the PTS digestion preformed the better identification for both proteins as well as peptides. Second, we applied the PTS digestion to all six GSCs cell lines. For each GSCs cell line, three time points were applied for protein extraction in the serum-induced differentiation or stem cell maintenance state, respectively. Third, all of samples were analyzed by reverse-phase-nanoLC/MS/MS, and the MS dataset were processed via MaxQuant followed by Persues. From MS-based proteomics, we have identified 7350 proteins and obtained 5329 quantified proteins. There are 224 were proteases such as ADAMs, MMPs, Caspases and Calpains, and these protease expressions were correlated with the several kinase networks such as TKR-MAPK-c-myc-CD44 etc. In conclusion, the results of this study provide an in-depth dynamic profiling of more than 200 proteases, and the next step is to classify the key regulators of proteases during differentiation.