**1. Introduction**

The incidence of central nervous system (CNS) has been increasing over the last 30 years, and unfortunately, become younger and younger [1]. More remarkable, patients (20–40%) with systemic cancer will occur metastatic disease to the CNS [2]. Primary malignant brain tumors and other metastatic disease to the CNS threaten human health. Astrocytoma is a type of primary malignant brain tumor that range from 20 to 40% of glioma, which is the most common

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

primary brain tumor in adults [3]. Here, we put research emphasis on two kinds of primary intracranial tumor, astrocytomas and pituitary adenomas. Astrocytomas cells possess the characteristic of high invasion to cause difficulty in therapy and high mortality (median survival = 9–12 months) [4]. A pituitary adenoma is quite common, and accounts for 10% of all primary intracranial neoplasias [5]. Even only a minority of pituitary adenomas (0.1–0.2%) develops into metastatic cancer, pituitary adenomas arise clinical problems: (a) Compress the adjacent brain organs appear symptoms including headache and visual failure. (b) An inappropriate hormone secretion led to hormone syndromes, including hypopituitarism, acromegaly, hyperprolactinemia and Cushing's syndrome [6]. The molecular mechanisms of those diseases remain unclear, and the traditional treatment models contain surgical excision, radiotherapy, and medical therapies [7]. Discovering in-depth molecular mechanisms, new diagnosis strategy, novel therapeutic targets are urgent.

Nitroproteomics methods were based on sample enrichment and mass spectrometry analysis. Modern nitroproteomics applies protein-separation-enrichment techniques such as gel methods and non-gel methods, including immunoprecipitation [11], anti-nitrotyrosine antibody-based enzyme-linked immunosorbent assay (ELISA) [27], and one/two-dimensional gel electrophoresis (1DGE/2DGE)-based Western blot analyses [9]. 1DGE/2DGE-based Western blots analyses can separate and preferentially enrich endogenous nitroproteins and also preliminarily determine the quantitative information of nitrotyrosine. Studies showed that the same protein was detected at multiple gel-spots on 2D electrophoresis gels, and single 2D electrophoresis gel-spot usually contains several proteins [28]. Therefore, 2D electrophoresis gel has advantages in protein component visualization, detection of protein species that are mainly derived from alternative splicing or PTMs [9]. Protein isoforms or variants present dynamic biological processes in vivo, and different protein species associated with different conditions and pathophysiological status [29]. We adopted 1DGE/2DGE-based Western blots analyses method: the scanned images of the silver-stained 2D electrophoresis gels and the visualization of Western blot membranes were input to a PDQuest system (Bio-Rad, version 7.1, Hercules, CA) to composite image that contained the Gaussian spots [30]. 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, it is also pointed out that the challenges faced by low abundance of tyrosine nitration and the elusive mass spectrometry result of a nitro group are existed [32]. For example, matrix-assisted laser desorption ionization (MALDI) is quite different from electrospray ionization (ESI)-MS when study the MS behaviors of a nitro-

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peptide [33]. Photochemical decompositions of the nitro group (–NO<sup>2</sup>

determine an amino acid sequence to study proteome and phosphoproteome.

energy laser decrease the precursor-ion intensity of a nitropeptide, making an MS spectrum much more complex in process of MALDI. However, the decomposition pattern of a nitropeptide was well clarified by the photochemical decomposition pattern ([M + H]+, [M + H – 16]+, [M + H –30]+, and [M + H – 32]+). Even ESI does not produce photochemical decompositions, scanning for the characteristic immonium ion (m/z 181.06) by the precursor ion could help accurately identify a nitropeptide or nitroprotein under ESI conditions [34]. Above key factors, including low abundance of nitroproteins, preferential enrichment methods, sensitivity of MS analysis, complicated MS behaviors of a nitro group, that all determine success or failure in the identity of in vivo nitroproteins. Thus, nitropeptide was detected by a vMALDI MS/MS method [9, 11]. Accumulation of MS/MS scans was used to increase the signal-to-noise ratio (S/N), which is needed for the detection of endogenous nitroproteins. A single MS/MS scan can

Protein tyrosine nitration is an (PTM) in nervous system tumor such as astrocytoma and pituitary adenomas, and participates in multiple complex biological processes [9, 11, 35, 36]. For human astrocytoma and pituitary adenomas nitroproteomics studies, 2DGE-based nitrotyrosine Western blot analysis with MALDI-TOF were used to identify endogenous nitroproteins and nitrotyrosine sites from human pituitary control and adenoma tissues, and 2DGE-based nitro-tyrosine Western blot analysis with liquid chromatography-electrospray ionization-quadrupole ion trap (LC-ESI-Q-IT) were used to identify endogenous nitroproteins and nitrotyrosine sites from human astrocytoma brain tissues. Bioinformatics and pathway analysis were used to determine domains/motifs in a nitroprotein, location of nitrotyrosine sites, and possible signaling pathways. A total of eight nitrotyrosine-containing proteins in human pituitary control tissues [9, 36], and nine nitroproteins and three nitroprotein-interacting

) induced by the high-

Protein tyrosine nitration is an important posttranslational modification (PTM) in nervous system tumors that is related with multiple abnormal pathophysiological processes [8]. The process of protein tyrosine nitration is formed from 3-nitrotyrosine group to position 3 of the tyrosine residue phenolic ring [9], which alters the electron density and pKa value (from ~10 for tyrosine into ~7.1 for 3-nitrotyrosine) of the tyrosine phenolic ring [10]. Such changes affect biochemical characteristics of the tyrosine residue that will change interaction between enzyme-substrate, antigen–antibody or receptor-ligand, when interacting regions were in nitration. Reactive nitrogen species (RNS) act as an important mediated material for protein nitration, and our studies [11] consistent with others [12, 13] have indicated that nitric oxide and protein nitration may play important roles in the nervous system tumors. Previous studies reported that (1) the inflammatory reaction is involved in nervous system tumors [14]; (2) NO and RNS are important inflammatory mediators [15]; and (3) increased production of NO, peroxinitrite and superoxide, occurs in nervous system tumors [16]; (4) higher levels of nitrotyrosine are observed in nervous system tumors than normal tissues with biochemical approaches and immunohistochemical, and only protein nitrotubulin and protein nitro-p53 have been determined in human nervous system tumors [17]. Furthermore, the amino acid analog 3-nitrotyrosine due to functional and morphological injury of mouse-neuroblastoma cell lines and rat-glioma cell lines [18]. These studies demonstrated the importance of protein tyrosine nitration in the pathogenesis of nervous system tumors. Illustrating the functions of nitroproteins might reveal in-depth molecular mechanisms and biological function of tyrosine nitration in human nervous system tumors. Literature-based review and comprehensive annotation of proteins on the SwissProt website were used to expound the nitroprotein domains/motifs, location of nitrotyrosine sites and possible signaling pathways relevant to nervous system tumors. Nitroproteins took part in multiple biological processes in the development of tumors as follows: (a) tumor cell migration and invasion [19]; (b) cell proliferation and apoptosis [20]; (c) chemotherapy resistance [21]; (d) signal transduction [22]; (e) phenotypic dedifferentiation [23]; (f) microtubule dynamic stabilization [24]; (g) tumor recurrence; (h) others such as immunoreaction and post-transcriptional regulation. Moreover, the discovery of tyrosine nitration being a reversible reaction [25] and having a competition between phosphorylation motif [26], led us to speculate that dynamic process of protein nitration might also be regulated and controlled. However, no definite target of intervention is found for tyrosine nitration in human nervous system tumors. It takes long time to study tumorrelated nitroproteins and to illustrate molecular mechanisms in tumor formation.

Nitroproteomics methods were based on sample enrichment and mass spectrometry analysis. Modern nitroproteomics applies protein-separation-enrichment techniques such as gel methods and non-gel methods, including immunoprecipitation [11], anti-nitrotyrosine antibody-based enzyme-linked immunosorbent assay (ELISA) [27], and one/two-dimensional gel electrophoresis (1DGE/2DGE)-based Western blot analyses [9]. 1DGE/2DGE-based Western blots analyses can separate and preferentially enrich endogenous nitroproteins and also preliminarily determine the quantitative information of nitrotyrosine. Studies showed that the same protein was detected at multiple gel-spots on 2D electrophoresis gels, and single 2D electrophoresis gel-spot usually contains several proteins [28]. Therefore, 2D electrophoresis gel has advantages in protein component visualization, detection of protein species that are mainly derived from alternative splicing or PTMs [9]. Protein isoforms or variants present dynamic biological processes in vivo, and different protein species associated with different conditions and pathophysiological status [29]. We adopted 1DGE/2DGE-based Western blots analyses method: the scanned images of the silver-stained 2D electrophoresis gels and the visualization of Western blot membranes were input to a PDQuest system (Bio-Rad, version 7.1, Hercules, CA) to composite image that contained the Gaussian spots [30]. 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, it is also pointed out that the challenges faced by low abundance of tyrosine nitration and the elusive mass spectrometry result of a nitro group are existed [32]. For example, matrix-assisted laser desorption ionization (MALDI) is quite different from electrospray ionization (ESI)-MS when study the MS behaviors of a nitropeptide [33]. Photochemical decompositions of the nitro group (–NO<sup>2</sup> ) induced by the highenergy laser decrease the precursor-ion intensity of a nitropeptide, making an MS spectrum much more complex in process of MALDI. However, the decomposition pattern of a nitropeptide was well clarified by the photochemical decomposition pattern ([M + H]+, [M + H – 16]+, [M + H –30]+, and [M + H – 32]+). Even ESI does not produce photochemical decompositions, scanning for the characteristic immonium ion (m/z 181.06) by the precursor ion could help accurately identify a nitropeptide or nitroprotein under ESI conditions [34]. Above key factors, including low abundance of nitroproteins, preferential enrichment methods, sensitivity of MS analysis, complicated MS behaviors of a nitro group, that all determine success or failure in the identity of in vivo nitroproteins. Thus, nitropeptide was detected by a vMALDI MS/MS method [9, 11]. Accumulation of MS/MS scans was used to increase the signal-to-noise ratio (S/N), which is needed for the detection of endogenous nitroproteins. A single MS/MS scan can determine an amino acid sequence to study proteome and phosphoproteome.

primary brain tumor in adults [3]. Here, we put research emphasis on two kinds of primary intracranial tumor, astrocytomas and pituitary adenomas. Astrocytomas cells possess the characteristic of high invasion to cause difficulty in therapy and high mortality (median survival = 9–12 months) [4]. A pituitary adenoma is quite common, and accounts for 10% of all primary intracranial neoplasias [5]. Even only a minority of pituitary adenomas (0.1–0.2%) develops into metastatic cancer, pituitary adenomas arise clinical problems: (a) Compress the adjacent brain organs appear symptoms including headache and visual failure. (b) An inappropriate hormone secretion led to hormone syndromes, including hypopituitarism, acromegaly, hyperprolactinemia and Cushing's syndrome [6]. The molecular mechanisms of those diseases remain unclear, and the traditional treatment models contain surgical excision, radiotherapy, and medical therapies [7]. Discovering in-depth molecular mechanisms, new

Protein tyrosine nitration is an important posttranslational modification (PTM) in nervous system tumors that is related with multiple abnormal pathophysiological processes [8]. The process of protein tyrosine nitration is formed from 3-nitrotyrosine group to position 3 of the tyrosine residue phenolic ring [9], which alters the electron density and pKa value (from ~10 for tyrosine into ~7.1 for 3-nitrotyrosine) of the tyrosine phenolic ring [10]. Such changes affect biochemical characteristics of the tyrosine residue that will change interaction between enzyme-substrate, antigen–antibody or receptor-ligand, when interacting regions were in nitration. Reactive nitrogen species (RNS) act as an important mediated material for protein nitration, and our studies [11] consistent with others [12, 13] have indicated that nitric oxide and protein nitration may play important roles in the nervous system tumors. Previous studies reported that (1) the inflammatory reaction is involved in nervous system tumors [14]; (2) NO and RNS are important inflammatory mediators [15]; and (3) increased production of NO, peroxinitrite and superoxide, occurs in nervous system tumors [16]; (4) higher levels of nitrotyrosine are observed in nervous system tumors than normal tissues with biochemical approaches and immunohistochemical, and only protein nitrotubulin and protein nitro-p53 have been determined in human nervous system tumors [17]. Furthermore, the amino acid analog 3-nitrotyrosine due to functional and morphological injury of mouse-neuroblastoma cell lines and rat-glioma cell lines [18]. These studies demonstrated the importance of protein tyrosine nitration in the pathogenesis of nervous system tumors. Illustrating the functions of nitroproteins might reveal in-depth molecular mechanisms and biological function of tyrosine nitration in human nervous system tumors. Literature-based review and comprehensive annotation of proteins on the SwissProt website were used to expound the nitroprotein domains/motifs, location of nitrotyrosine sites and possible signaling pathways relevant to nervous system tumors. Nitroproteins took part in multiple biological processes in the development of tumors as follows: (a) tumor cell migration and invasion [19]; (b) cell proliferation and apoptosis [20]; (c) chemotherapy resistance [21]; (d) signal transduction [22]; (e) phenotypic dedifferentiation [23]; (f) microtubule dynamic stabilization [24]; (g) tumor recurrence; (h) others such as immunoreaction and post-transcriptional regulation. Moreover, the discovery of tyrosine nitration being a reversible reaction [25] and having a competition between phosphorylation motif [26], led us to speculate that dynamic process of protein nitration might also be regulated and controlled. However, no definite target of intervention is found for tyrosine nitration in human nervous system tumors. It takes long time to study tumor-

related nitroproteins and to illustrate molecular mechanisms in tumor formation.

diagnosis strategy, novel therapeutic targets are urgent.

108 Electrophoresis - Life Sciences Practical Applications

Protein tyrosine nitration is an (PTM) in nervous system tumor such as astrocytoma and pituitary adenomas, and participates in multiple complex biological processes [9, 11, 35, 36]. For human astrocytoma and pituitary adenomas nitroproteomics studies, 2DGE-based nitrotyrosine Western blot analysis with MALDI-TOF were used to identify endogenous nitroproteins and nitrotyrosine sites from human pituitary control and adenoma tissues, and 2DGE-based nitro-tyrosine Western blot analysis with liquid chromatography-electrospray ionization-quadrupole ion trap (LC-ESI-Q-IT) were used to identify endogenous nitroproteins and nitrotyrosine sites from human astrocytoma brain tissues. Bioinformatics and pathway analysis were used to determine domains/motifs in a nitroprotein, location of nitrotyrosine sites, and possible signaling pathways. A total of eight nitrotyrosine-containing proteins in human pituitary control tissues [9, 36], and nine nitroproteins and three nitroprotein-interacting proteins in a human nonfunctional pituitary adenoma tissue [11], 18 nitroproteins and their 23 nitrotyrosine sites in human astrocytoma tissues [35], were identified with 2DE-MS/MS. The nitration site was located onto the corresponding functional domain, and each nitroprotein was carried out pathway analysis to speculate the possible biological function.

MALDI-MS/MS: The immunopositive 2D gel spots were excised, digested, purified, eluted, airdried, redissolved, and were spots onto MALDI-plate, which were subjected to MALDI-MS/

MS/MS data were used to identify the protein and nitrotyrosine sites by searching the SwissProt and NCBInr databases with SQUEST or Mascot software, with mass modifica-

Met. Protein domains and motifs analyses were carried out with ScanProsite software (http:// us.expasy.org/tools/scanprosite). Nitrotyrosine sites within a given domain or motif were determined with MotifScan software (http://myhits.isb-sib.ch/cgi-bin/motif\_scan). The functions and experimental data-based model of nitroproteins was searched on the Swiss-Prot

Literature-based bioinformatics and comprehensive annotation of protein in the SwissProt page were used to rationalize the functional characteristics of each nitroprotein, and to provide important clues to the biological significance of each nitroprotein relevant to tumors.

One1D gel-based Western blotting in combination with anti-nitrotyrosine antibody analysis revealed that the overall level of protein tyrosine nitration in astrocytoma was significantly

The challenges faced by low abundance of tyrosine nitration and the elusive mass spectrometry result of a nitro group are existed. Modern nitroproteomics applies protein-separation-enrichment techniques such as gel methods and non-gel methods, including immunoprecipitation [11, 37], anti-nitrotyrosine antibody-based enzyme-linked immunosorbent assay (ELISA) [27], and one/two-dimensional gel electrophoresis (1DGE/2DGE)-based Western blot analyses [9, 35]. 1DGE/2DGE-based Western blots analyses can separate and preferentially enrich endogenous nitroproteins and also preliminarily determine the quantitative information of nitrotyrosine. It should not be neglected that limitations of 2DGE-based method including coverage of proteome, dynamic range, sensitivity and throughput, which always were restricted by the amount of samples. Also, the non-nitrated tryptic peptides are much more than nitrated tryptic peptides after a 2DGE-separated nitroprotein was digested, which will interrupt MS/MS signal of nitrated tryptic peptides [38]. However, for the proteome study, a 2DGE gel could detect more than 1000 spots [39]. Different proteins may be contained within the same spot [40].

COCH<sup>2</sup>

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– H) at Cys, +16 Da (oxidation) at

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MS analysis [37]. The detailed procedure has been described [9, 11, 36].

– H) at Tyr, +57 Da (+NH<sup>2</sup>

database annotation page and the related literature resources.

**2.5. Annotation of functional characteristics of the nitroproteins**

**3.1. The level of protein tyrosine nitration in nervous system tumors**

**3.2. Enrichment of endogenous nitroproteins in nervous system tumors**

**2.4. Identification of nitroproteins with MS/MS**

tions of +45 Da (+NO<sup>2</sup>

**3. Results and discussions**

higher than the controls (**Figure 1**).
