**1. Introduction**

Glioblastoma (GBM) is known to be the most common and aggressive brain tumour in adults. Despite the enormous efforts to overcome this tumour for many years, the median survival for GBM patients remains around only 1 year [1]. GBM is characterized by high invasiveness

and intratumoral heterogeneity (ITH) [2, 3]. Up to date, it is known that GBM‐ITH contributes to the resistance to chemotherapy, radiation and surgical resection. Since functional diversity is the main feature of multilineage differentiation of cancer stem cells (CSCs) [4, 5], glioblastoma stem cells (GSCs) were thought to be major therapeutic targets of GBM. Furthermore, post‐translational modifications (PTMs) of GSCs are reported to tightly regulate highly tumourigenic potential of GSCs through aberrant signalling [6, 7]. Therefore, it is important to comprehensively elucidate PTM‐based GSC signalling networks for developing the effective treatment of GBM.

Advanced nanoscale liquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS) enables us to identify and quantify thousands of proteins in a single experiment [8]. Moreover, using the nanoLC‐MS/MS system coupled to the high‐affinity enrichment methods of the peptides with PTMs, we can also acquire in‐depth biological information on PTM dynamics. In this chapter, we introduce high‐resolution shotgun proteomics technology for large‐scale PTM determination in combination with statistical bioinformatics platforms such as IPA [9], NetworKIN [10, 11] and PTMapper [12].
