**3. Results and discussions**

### **3.1. The level of protein tyrosine nitration in nervous system 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 higher than the controls (**Figure 1**).

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

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].

A similar 2DGE-based Western blot coupled with anti-nitrotyrosine antibody was used to analyze nitroproteins in human pituitary tissues [35, 38]. Each pituitary silver-stained 2D gel image (pI 3–10; Mr. 10–100 kDa) contained ca. 1000 protein spots, and a total of 32 nitrotyro-

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An immunoprecipitation, nitrotyrosine affinity column (NTAC)-based MS/MS approach was also used to enrich and identify nitroproteins from a pituitary adenoma tissue (**Figure 3**) [37]. Briefly, the NTAC was prepared with protein G beads cross-linked with anti-nitrotyrosine antibodies. A volume (600 μl) of protein extracts from 62 mg wet weight of a pituitary adenoma

**Figure 2.** 2DGE-based Western blot analysis of nitroproteins in an IV-grade astrocytoma tissue (500 μg protein per 2D gel). (A) Coomassie blue-stained 2DGE image (before transfer of proteins). (B) Coomassie blue-stained 2DGE image (after transfer of proteins). (C) Western blotting image of nitroproteins (anti-nitrotyrosine antibodies + secondary antibodies). (D) Negative control of Western blotting to show the cross-reaction of the secondary antibody (only the secondary antibody; no anti-nitrotyrosine antibody). Reproduced from Peng and Zhan [35], with permission from

Springer, copyright 2015.

sine immunopositive Western blot spots were detected in human pituitary tissues.

**Figure 1.** The nitrotyrosine immunoactivities in different grade (I, II, III, and IV) of astrocytoma tissues relative to normal controls (N), detected with nitrotyrosine immunoaffinty-based western blotting (n = 3). Tumor = sum of different grade (I, II, III, and IV) of astrocytomas. \*p < 0.05. Reproduced from Peng and Zhan [35], with permission from Springer, copyright 2015.

Many proteins were detected in several analyzed spots showing 2DE-MS separate ability at the protein species level [43]. Indeed, higher sensitivity had been expected when 2DE coupled with high-sensitivity LC–MS, and will detect, identify and quantify human proteome nearly 500,000 (with an estimated resolution) protein species [28]. We adopted 2DGE-based Western blots analyses method to separate and detect nitroproteins in nervous system tumors. Our previous study obtained enrichment of endogenous nitroproteins in human pituitary [9, 36] and astrocytoma [35].

The 2DGE-based Western blot coupled with anti-nitrotyrosine antibody was used to analyze nitroproteins in human astrocytoma tissues (**Figure 2**) [17]. Nearly 1100 protein spots were detected in each Coomassie-stained 2D gel. Most proteins were distributed within a range of pI 4–8 and Mr. of 15–150 kDa. A total of 57 nitrotyrosine-immunopositive gel spots were detected, and each positive spot corresponded to a Coomassie-stained 2D gel spot. In order to show non-specific nitrotyrosine-immunopositive Western blot spots, we setup a control experiment without primary antibody to determine cross-reactivity of the secondary antibody.

A similar 2DGE-based Western blot coupled with anti-nitrotyrosine antibody was used to analyze nitroproteins in human pituitary tissues [35, 38]. Each pituitary silver-stained 2D gel image (pI 3–10; Mr. 10–100 kDa) contained ca. 1000 protein spots, and a total of 32 nitrotyrosine immunopositive Western blot spots were detected in human pituitary tissues.

An immunoprecipitation, nitrotyrosine affinity column (NTAC)-based MS/MS approach was also used to enrich and identify nitroproteins from a pituitary adenoma tissue (**Figure 3**) [37]. Briefly, the NTAC was prepared with protein G beads cross-linked with anti-nitrotyrosine antibodies. A volume (600 μl) of protein extracts from 62 mg wet weight of a pituitary adenoma

Many proteins were detected in several analyzed spots showing 2DE-MS separate ability at the protein species level [43]. Indeed, higher sensitivity had been expected when 2DE coupled with high-sensitivity LC–MS, and will detect, identify and quantify human proteome nearly 500,000 (with an estimated resolution) protein species [28]. We adopted 2DGE-based Western blots analyses method to separate and detect nitroproteins in nervous system tumors. Our previous study obtained enrichment of endogenous nitroproteins in human pituitary [9, 36]

**Figure 1.** The nitrotyrosine immunoactivities in different grade (I, II, III, and IV) of astrocytoma tissues relative to normal controls (N), detected with nitrotyrosine immunoaffinty-based western blotting (n = 3). Tumor = sum of different grade (I, II, III, and IV) of astrocytomas. \*p < 0.05. Reproduced from Peng and Zhan [35], with permission from Springer,

The 2DGE-based Western blot coupled with anti-nitrotyrosine antibody was used to analyze nitroproteins in human astrocytoma tissues (**Figure 2**) [17]. Nearly 1100 protein spots were detected in each Coomassie-stained 2D gel. Most proteins were distributed within a range of pI 4–8 and Mr. of 15–150 kDa. A total of 57 nitrotyrosine-immunopositive gel spots were detected, and each positive spot corresponded to a Coomassie-stained 2D gel spot. In order to show non-specific nitrotyrosine-immunopositive Western blot spots, we setup a control experiment without primary antibody to determine cross-reactivity of the secondary

and astrocytoma [35].

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copyright 2015.

antibody.

**Figure 2.** 2DGE-based Western blot analysis of nitroproteins in an IV-grade astrocytoma tissue (500 μg protein per 2D gel). (A) Coomassie blue-stained 2DGE image (before transfer of proteins). (B) Coomassie blue-stained 2DGE image (after transfer of proteins). (C) Western blotting image of nitroproteins (anti-nitrotyrosine antibodies + secondary antibodies). (D) Negative control of Western blotting to show the cross-reaction of the secondary antibody (only the secondary antibody; no anti-nitrotyrosine antibody). Reproduced from Peng and Zhan [35], with permission from Springer, copyright 2015.

[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;

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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)

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

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

(3) a prepared sample could be reanalyzed in several weeks.

that is derived from sorcin (C9J0K6) in human astrocytoma tissue (**Figure 4**).

nitration of sorcin in astrocytoma is obviously higher than the control tissues.

**3.4. Functional characteristics of the nitroproteins**

as well as the immune system.

**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 Science, copyright 2006.

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–protein complexes, followed by tripsin digestion and MS/MS identification.
