**2. System‐wide proteomic analysis of PTM dynamics**

PTMs are widely known to play crucial roles in cell fate control, such as proliferation, differentiation and apoptosis. More than 500 kinds of PTMs regarding eukaryotes and prokaryotes have been registered with Unimod, a comprehensive database of protein modifications for mass spectrometry [13]. Recent technological advances in mass spectrometry‐based proteomics in combination with appropriate enrichment techniques for each PTM enable us to perform comprehensive identification and quantification of PTMs [14]. Here, we introduce biochemical purification methods for highly sensitive detection of the representative PTMs: phosphorylation, acetylation and ubiquitination (**Figure 1**).

**2.3. Ubiquitination**

acetylation, Ub: ubiquitination, TiO<sup>2</sup>

**GSCs**

The ubiquitin system transmits protein degradation signal to proteasome as well as regulates multiple cellular functions such as cell‐cycle progression, DNA repair and transcriptional regulation. Dysfunction of this system leads to various pathological conditions [24]. Ubiquitination sites are detected as diglycine (Gly‐Gly) remnants on the modified lysine resi-

**Figure 1.** Strategy for mass spectrometry‐based identification of peptides modified with phosphorylation, acetylation and ubiquitination. Regarding ubiquitinated lysine residues, Gly‐Gly remnants are generated from the C‐terminal of ubiquitin as a consequence of tryptic digestion. PTMs: post‐translational modifications, P: phosphorylation, Ac:

Comprehensive Network Analysis of Cancer Stem Cell Signalling through Systematic...

http://dx.doi.org/10.5772/intechopen.69647

267

dues, which are generated by tryptic digestion of ubiquitinated proteins [25, 26].

: titanium dioxide.

**3. Systematic characterization of the phosphoproteome dynamics in** 

The quantitative information on the phosphoproteome dynamics can provide us with systematic description of the key machinery for cellular signalling. In this section, we introduce two examples of global phosphoproteome analyses of GSCs using SILAC (stable isotope labelling by amino acids in cell culture)‐based quantitative technique [27, 28] (**Figure 2**). One was carried out using epidermal growth factor (EGF) to elucidate the mechanism for stemness maintenance of GSCs [29], whereas the other was conducted through serum‐induced differentiation of GSCs to unveil the key pathways responsible for disrupting stemness characteristics [30].

#### **2.1. Phosphorylation**

Protein phosphorylation is recognized as one of the most important and well‐studied PTMs and regulates a variety of biological processes by transmitting diverse external signals [15, 16]. About as many as 280,000 phosphorylation sites have already been registered in PhosphoSitePlus, a knowledgebase containing non‐redundant mammalian PTMs [17]. Titanium dioxide (TiO<sup>2</sup> ), which has very high affinity for phosphorylated peptides, is widely used for large‐scale phosphoproteome analysis [18, 19].

#### **2.2. Acetylation**

Lysine acetylation plays a key role in modulating transcriptional regulation through the coordinated function of histone acetyltransferases (HATs) and histone deacetylases (HDACs) [20]. The stabilization of p53, one of the most important transcription factors, is reported to greatly depend on lysine acetylation [21]. Thousands of lysine acetylation sites can be identified using an antibody against acetyl‐lysine in combination with a high‐resolution mass spectrometry system [22, 23].

Comprehensive Network Analysis of Cancer Stem Cell Signalling through Systematic... http://dx.doi.org/10.5772/intechopen.69647 267

**Figure 1.** Strategy for mass spectrometry‐based identification of peptides modified with phosphorylation, acetylation and ubiquitination. Regarding ubiquitinated lysine residues, Gly‐Gly remnants are generated from the C‐terminal of ubiquitin as a consequence of tryptic digestion. PTMs: post‐translational modifications, P: phosphorylation, Ac: acetylation, Ub: ubiquitination, TiO<sup>2</sup> : titanium dioxide.

#### **2.3. Ubiquitination**

),

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 effec-

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],

PTMs are widely known to play crucial roles in cell fate control, such as proliferation, differentiation and apoptosis. More than 500 kinds of PTMs regarding eukaryotes and prokaryotes have been registered with Unimod, a comprehensive database of protein modifications for mass spectrometry [13]. Recent technological advances in mass spectrometry‐based proteomics in combination with appropriate enrichment techniques for each PTM enable us to perform comprehensive identification and quantification of PTMs [14]. Here, we introduce biochemical purification methods for highly sensitive detection of the representative PTMs:

Protein phosphorylation is recognized as one of the most important and well‐studied PTMs and regulates a variety of biological processes by transmitting diverse external signals [15, 16]. About as many as 280,000 phosphorylation sites have already been registered in PhosphoSitePlus, a knowledgebase containing non‐redundant mammalian PTMs [17]. Titanium dioxide (TiO<sup>2</sup>

which has very high affinity for phosphorylated peptides, is widely used for large‐scale phos-

Lysine acetylation plays a key role in modulating transcriptional regulation through the coordinated function of histone acetyltransferases (HATs) and histone deacetylases (HDACs) [20]. The stabilization of p53, one of the most important transcription factors, is reported to greatly depend on lysine acetylation [21]. Thousands of lysine acetylation sites can be identified using an antibody against acetyl‐lysine in combination with a high‐resolution mass spectrometry

tive treatment of GBM.

**2.1. Phosphorylation**

**2.2. Acetylation**

system [22, 23].

phoproteome analysis [18, 19].

NetworKIN [10, 11] and PTMapper [12].

**2. System‐wide proteomic analysis of PTM dynamics**

266 Applications of RNA-Seq and Omics Strategies - From Microorganisms to Human Health

phosphorylation, acetylation and ubiquitination (**Figure 1**).

The ubiquitin system transmits protein degradation signal to proteasome as well as regulates multiple cellular functions such as cell‐cycle progression, DNA repair and transcriptional regulation. Dysfunction of this system leads to various pathological conditions [24]. Ubiquitination sites are detected as diglycine (Gly‐Gly) remnants on the modified lysine residues, which are generated by tryptic digestion of ubiquitinated proteins [25, 26].
