**3.3. MS/MS identification of nitroproteins and nitrated sites in nervous system tumors**

MS/MS is the mainstream technique to identify protein species and PTMs products with verification of amino acid sequence, splicing sites, and modification site [31]. However, the very low abundance of tyrosine nitration in a proteome (one nitration in ~10<sup>6</sup> tyrosine residues) and the complicated mass spectrometry behaviors of a nitro group that we studied in nitroproteins complicate analyses [34, 38]. Different mass spectrometry has different advantages and complicated mass spectrometry behaviors, so choosing the proper method or combination with each other is necessary. For example, the MS behaviors of a nitropeptide differ significantly between matrix-assisted laser desorption ionization (MALDI)-and electrospray ionization (ESI)-MS on process of photochemical decompositions of the nitro group (–NO<sup>2</sup> ) [33, 41]. Additionally, Specificity and sensitivity of the MALDI–LTQ MS/MS analytical system to characterize each nitroprotein and nitroprotein–protein complex should be considered. According to our previous experience [42], MALDI–LTQ has a number of advantages: (1) highly sensitive; (2) high accuracy measurement on amino acid sequence and nitration sites; (3) a prepared sample could be reanalyzed in several weeks.

For human astrocytoma and pituitary adenomas nitroproteomics studies, 2DGE-based nitrotyrosine Western blot analysis with MALDI-TOF were used to obtain endogenous nitroproteins from human pituitary control and adenoma tissues, and 2DGE-based nitrotyrosine Western blot analysis with liquid chromatography-electrospray ionization-quadrupole ion trap (LC-ESI-Q-IT) were used to obtain endogenous nitroproteins from human astrocytoma brain tissues, flowed by identification of nitroproteins, proteins interacted with nitroproteins and nitrotyrosine sites. A representative MS/MS spectrum was shown to identify nitropeptide (ITFDDnYIAC\*C\*VK) that is derived from sorcin (C9J0K6) in human astrocytoma tissue (**Figure 4**).

A total of eight nitroproteins was identified in human normal pituitary tissues with 2DGE-MS/ MS [9, 36], and nine nitroproteins were identified in a human nonfunctional pituitary adenoma tissue with NTAC-MS/MS [11], and 18 nitroproteins and their 20 nitrotyrosine sites was identified in a human astrocytoma tissue with 2DGE-MS/MS [35] (**Table 1**). Three nitroprotein–protein complexes were also identified in a human nonfunctional pituitary adenoma tissue: the nitrated beta-subunit of cAMP-dependent protein kinase (PKA) complex, the nitrated proteasome–ubiquitin complex, and the nitrated interleukin 1 family member 6–interleukin 1 receptor–interleukin 1 receptor-associated kinase-like 2 (IL1-F6–IL1-R–IRAK-2) complex [37].

Furthermore, the 2DE-MS/MS-identified nitrosorcin in astrocytoma tissues was confirmed with immunoprecipitation coupled with 1D gel-Western blot experiments (**Figure 5**). The tyrosine nitration of sorcin was measured by immunoprecipitation coupled with Western blotting between IV-grade astrocytoma and normal control (N) tissues. Thus overall status of tyrosine nitration in the whole tissues could be displayed with anti-nitrotyrosine antibody. The results confirmed the tyrosine nitration of sorcin in astrocytoma, and the level of tyrosine nitration of sorcin in astrocytoma is obviously higher than the control tissues.

### **3.4. Functional characteristics of the nitroproteins**

tissue was diluted (1,1, v/v) with binding/washing buffer. Then, 500 μl diluted sample was incubated with the prepared NTAC to enrich and isolate nitroproteins and nitroprotein–pro-

**Figure 3.** The use of NTAC to characterize nitroproteins and their complexes. A parallel control experiment was carried out without any anti-3-nitrotyrosine antibody. Reproduced from Zhan and Desiderio [11], with permission from Elsevier

MS/MS is the mainstream technique to identify protein species and PTMs products with verification of amino acid sequence, splicing sites, and modification site [31]. However, the very

and the complicated mass spectrometry behaviors of a nitro group that we studied in nitroproteins complicate analyses [34, 38]. Different mass spectrometry has different advantages and complicated mass spectrometry behaviors, so choosing the proper method or combination with each other is necessary. For example, the MS behaviors of a nitropeptide differ significantly between matrix-assisted laser desorption ionization (MALDI)-and electrospray ionization (ESI)-MS on process of photochemical decompositions of the nitro group (–NO<sup>2</sup>

tyrosine residues)

)

tein complexes, followed by tripsin digestion and MS/MS identification.

low abundance of tyrosine nitration in a proteome (one nitration in ~10<sup>6</sup>

**tumors**

Science, copyright 2006.

114 Electrophoresis - Life Sciences Practical Applications

**3.3. MS/MS identification of nitroproteins and nitrated sites in nervous system** 

Comprehensive analysis of the functional characteristics of those nine nitroproteins and three nitroproten-protein complexes in a pituitary adenoma biological system revealed several important functional pathways involved in protein tyrosine nitration (**Figure 6**): Nitrated RHOGAP5 and nitrated rhophilin 2 are involved in the GTPase signal pathway. Nitrated CENT-beta 1 and nitrated PKAR1-beta are involved in the PKA signal pathway. IRAK-2 in the IL1-R complex and nitrated IL1-F6 are involved in the cytokine system. The nitrated proteasome–ubiquitin complex is an important enzymatic complex involved in the intracellular nonlysosomal proteolytic pathway. Nitrated LIRA4 might be involved in the immune system. Nitrated ZFP432 is involved in transcription regulatory systems. The nitrated S1P lyase 1 participates in sphingolipid metabolism to regulate cell proliferation, survival, and cell death as well as the immune system.

Comprehensive analysis of the functional characteristics of 18 astrocytoma nitroproteins revealed those nitroproteins participated in multiple cancer-related biological processes (**Figure 7**): (a) microtubule dynamic stabilization and cytoprotection, which mainly involvedβVIII-tubulin, and β-tubulin; (b) tumor migration and metastasis, which mainly

**Figure 4.** MS/MS spectrum of a nitropeptide (ITFDDnYIAC\*C\*VK) that is derived from sorcin (C9J0K6) in astrocytoma tissue. nY = nitrotyrosine residue. Reproduced from Peng and Zhan [35], with permission from Springer, copyright 2015.


involved sorcin, isoform 2 of Toll-like receptor 9, isoform 2 of Arf-GAP with SH3 domain/ ANK repeat and PH domain-containing protein 3, transducin-like enhancer protein 2, and GPR52; (c) chemotherapy resistance, which mainly involved Ras-related protein Rab 8,

Note: nY = nitrotyrosine. Pituitary adenoma and control modified from Zhan and Desiderio [9, 11, 36], with permission from Elsevier Science, copyright 2004, 2006 and 2007. Astrocytoma modified from Peng and Zhan [35], with permission

General transcription factor 3C polypeptide 3 (Fragment)

domain-containing protein 2 (O43150–2)

**Table 1.** Nitroproteins and non-nitrated proteins identified from nervous system tumor.

Isoform 2 of Arf-GAP with SH3 domain, ANK repeat and PH

Isoform 2 of Toll-like receptor 9 (Q9NR96–2) nY419 Transducin-like enhancer protein 2 (B4DE62) nY369

**Nitrated protein name nY site**

The Use of Gel Electrophoresis and Mass Spectrometry to Identify Nitroproteins in Nervous…

Mitochondrial co-chaperone protein HscB [Q8IWL3] nY<sup>128</sup> Actin [P03996] (ACTA2, ACTG2, ACTC1) nY<sup>296</sup> Synaptosomal-associated protein (SNAP91) nY237 Ig alpha Fc receptor [P24071] (FCAR) nY223

PKG 2 [Q13237] (PRKG2) nY354 Stanniocalcin 1[P52823] (STC1) nY<sup>159</sup>

Regulating synaptic membrane exocytosis protein 1 (Q86UR5) nY<sup>926</sup> Isoform 2 of Grainyhead-like protein 1 homolog (Q9NZI5–2) nY400, nY402 Probable G-protein coupled receptor 52 (Q9Y2T5) nY<sup>281</sup>, nY284 Sorcin (C9J0K6) nY<sup>116</sup> Tubulin beta chain (P07437) nY106

Tubulin beta-2B chain (Q9BVA1) nY106 Tubulin beta-3 chain (Q13509) nY106 Ig kappa chain V-I region WAT (P80362) nY49 Coiled-coil domain-containing protein 105 (Q8IYK2) nY372 Helicase ARIP4 (E7EU19) nY407

Tubulin beta-2A chain (Q13885) nY106, nY183, nY200

Isoform 2 of Signal-induced proliferation-associated 1-like protein

nY33

http://dx.doi.org/10.5772/intechopen.76889

117

nY1369, nY1387

nY110

nY<sup>75</sup>

Progestin and adipoQ receptor family member III [Q6TCH7]

Astrocytoma Ras-related protein Rab-8B (H0YMN7) nY<sup>77</sup>;nY<sup>78</sup>

Pituitary control Proteasome subunit alpha type 2 (PSMA2) nY<sup>228</sup>

(PAQR3)

2 (Q9P2F8–2)

(H7C0C0)

from Springer, copyright 2015.

**Nervous system tumors/**

**control**


Comprehensive analysis of the functional characteristics of 18 astrocytoma nitroproteins revealed those nitroproteins participated in multiple cancer-related biological processes (**Figure 7**): (a) microtubule dynamic stabilization and cytoprotection, which mainly involvedβVIII-tubulin, and β-tubulin; (b) tumor migration and metastasis, which mainly

**Nitrated protein name nY site**

**Figure 4.** MS/MS spectrum of a nitropeptide (ITFDDnYIAC\*C\*VK) that is derived from sorcin (C9J0K6) in astrocytoma tissue. nY = nitrotyrosine residue. Reproduced from Peng and Zhan [35], with permission from Springer, copyright 2015.

> Leukocyte immunoglobulin-like receptor A4 [P59901] nY404 Zinc finger protein 432 [O94892] nY41 PKA beta regulatory subunit [P31321] (PRKAR1B) nY20 Sphingosine-1-phosphate lyase 1 [O95470] nY356, Y366 Centaurin beta 1 [Q15027] nY485 Proteasome subunit alpha type 2 [P25787] (PSMA2) nY<sup>228</sup> Interleukin 1 family member 6 [Q9UHA7] (IL1F6) nY<sup>96</sup> Rhophilin 2 [Q8IUC4] (RHPN2) nY<sup>258</sup>

Pituitary adenoma Rho-GTPase-activating 5 [Q13017] (ARHGAP5) nY550

**Nervous system tumors/**

116 Electrophoresis - Life Sciences Practical Applications

**control**

Note: nY = nitrotyrosine. Pituitary adenoma and control modified from Zhan and Desiderio [9, 11, 36], with permission from Elsevier Science, copyright 2004, 2006 and 2007. Astrocytoma modified from Peng and Zhan [35], with permission from Springer, copyright 2015.

**Table 1.** Nitroproteins and non-nitrated proteins identified from nervous system tumor.

involved sorcin, isoform 2 of Toll-like receptor 9, isoform 2 of Arf-GAP with SH3 domain/ ANK repeat and PH domain-containing protein 3, transducin-like enhancer protein 2, and GPR52; (c) chemotherapy resistance, which mainly involved Ras-related protein Rab 8,

**Figure 5.** Nitrotyrosine immune activities in nitrosorcin-immunoprecipitated products from IV-astrocytoma and normal control (N) tissues. Reproduced from Peng and Zhan [35], with permission from springer, copyright 2015.

βIII-tubulin, βIVa-tubulin, βVI-tubulin, and sorcin; (d) cell proliferation and apoptosis, which mainly involved а-tubulin, isoform 2 of Grainyhead-like protein 1, Ras-related protein Rab 8, regulating synaptic membrane exocytosis protein 1, and coiled-coil domain-containing protein 105; (e) phenotypic dedifferentiation, which involved gamma-tubulin; (f) signal transduction, which mainly involved isoform 2 of signal-induced proliferation-associated 1-like protein 2; (g) others such as transcription, immune response, and transformation; (h) tumor

**Figure 7.** Experimental data-based diagram that rationalizes nitrotyrosine-containing proteins in the glioma biological

The Use of Gel Electrophoresis and Mass Spectrometry to Identify Nitroproteins in Nervous…

http://dx.doi.org/10.5772/intechopen.76889

119

system. Reproduced from Peng and Zhan [35], with permission from Springer, copyright 2015.

Protein tyrosine nitration, as one of an important PTMs generated in nervous system tumor, participates in multiple complex biological processes, including cell proliferation and apoptosis, metastasis, migration, drug-resistance, cytoskeleton, signal transduction, immune response and cellular differentiation [43]. What is the mechanism for protein tyrosine nitration in carcinogenesis and development of malignant tumors? How to identify the protein targets and exact modified sites of tyrosine nitration? One/two-dimensional gel electrophoresis-based nitrotyrosine Western blot analysis and tandem mass spectrometry have been suc-

Further study is needed to solve those limits on protein nitration: (1) Protein tyrosine nitration and heterogeneity of neoplasm. (2) Three-dimensional spatial structure of a nervous system tumor-related nitroprotein. (3) Consistency issues between body fluid and tissue. With the clarification of those issues on nitroproteins, protein tyrosine nitration will have a significant

malignancy and recurrence-free survival, which involvedβII-tubulin and βIII-tubulin.

cessfully applied in the analysis of nitroproteins in nervous system tumors.

impact on the field of nervous system tumors.

**4. Conclusion(s)**

**Figure 6.** Experimental data-based model of nitroproteins and their functions in human nonfunctional pituitary adenomas. NO<sup>2</sup> − , nitroprotein. Reproduced from Zhan and Desiderio [11], with permission from Elsevier Science, copyright 2006.

The Use of Gel Electrophoresis and Mass Spectrometry to Identify Nitroproteins in Nervous… http://dx.doi.org/10.5772/intechopen.76889 119

**Figure 7.** Experimental data-based diagram that rationalizes nitrotyrosine-containing proteins in the glioma biological system. Reproduced from Peng and Zhan [35], with permission from Springer, copyright 2015.

βIII-tubulin, βIVa-tubulin, βVI-tubulin, and sorcin; (d) cell proliferation and apoptosis, which mainly involved а-tubulin, isoform 2 of Grainyhead-like protein 1, Ras-related protein Rab 8, regulating synaptic membrane exocytosis protein 1, and coiled-coil domain-containing protein 105; (e) phenotypic dedifferentiation, which involved gamma-tubulin; (f) signal transduction, which mainly involved isoform 2 of signal-induced proliferation-associated 1-like protein 2; (g) others such as transcription, immune response, and transformation; (h) tumor malignancy and recurrence-free survival, which involvedβII-tubulin and βIII-tubulin.
