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

从GBM细胞系中建立的6个GSC克隆用于基于MS的蛋白质组学研究。我们首先检测了尿素或相转移表面活性物质(PTS)辅助的胰酶消化中鉴定出的蛋白质数量，得出结论：PTS消化对蛋白质和多肽都具有更好的鉴定效果。其次，我们将PTS消化应用于所有六个GSCs细胞系。对于每个GSCs细胞系，分别在血清诱导分化或干细胞维持状态下应用三个时间点进行蛋白质提取。用反相纳米LC/MS/MS对样品进行分析，用MaxQuant和Persues对MS数据集进行处理。从基于MS的蛋白质组学中，我们已经鉴定了7350个蛋白质，获得了5329个定量蛋白质。有224种蛋白水解酶的表达与TKR-MAPK-c-myc-CD44等几个激酶网络相关。综上所述，本研究提供了200多种蛋白水解酶的动态图谱，下一步是对分化过程中的关键蛋白水解酶进行分类。

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.