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Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum.

Uda M, Kawasaki H, Shigenaga A, Baba T, Yamakura F - Biosci. Rep. (2012)

Bottom Line: We also observed that the total amount of NO2Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05).The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus.This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.

View Article: PubMed Central - PubMed

Affiliation: Sportology Center, Juntendo University Graduate School of Medicine, Chiba, Japan.

ABSTRACT
Nitration of tryptophan residues is a novel post-translational modification. In the present study, we examined whether NO2Trp (nitrotryptophan)-containing proteins are produced in the hippocampus and cerebellum of the adult rat under physiological conditions in vivo. Using Western blot analysis with anti-6-NO2Trp-specific antibody, we found many similar immunoreactive spots in the protein extracts from both regions. These spots were subsequently subjected to trypsin digestion and LC-ESI-MS/MS (LC-electrospray ionization-tandem MS) analysis. We identified several cytoskeletal proteins and glycolytic enzymes as NO2Trp-containing proteins and determined the position of nitrated tryptophan residues with significant ion score levels (P<0.05) in several proteins in both regions. We also observed that the total amount of NO2Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05). Moreover, IP (immunoprecipitation) assays using anti-aldolase C antibody showed that the relative intensity of immunostaining for NO2Trp over aldolase C was much higher in cerebellum than in hippocampus. The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus. This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.

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Related in: MedlinePlus

Representative results of Western blotting with an anti-6-NO2Trp antibody andSypro Ruby-stained gel after two-dimensional electrophoresis(A) and (C) are Western blotting and Sypro Ruby gel staining ofhippocampus respectively. (B) and (D) are those of cerebellum. Opencircled spots and spots indicated by arrows in hippocampus (h1–h16) and cerebellum(c1–c15) were subjected to LC-ESI-MS/MS analysis.
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Figure 1: Representative results of Western blotting with an anti-6-NO2Trp antibody andSypro Ruby-stained gel after two-dimensional electrophoresis(A) and (C) are Western blotting and Sypro Ruby gel staining ofhippocampus respectively. (B) and (D) are those of cerebellum. Opencircled spots and spots indicated by arrows in hippocampus (h1–h16) and cerebellum(c1–c15) were subjected to LC-ESI-MS/MS analysis.

Mentions: Figure 1 shows representative results of Western blottingwith the anti-6-NO2Trp antibody (Figures 1A and1B) and Sypro Ruby staining gels (Figures 1C and 1D) after two-dimensional PAGE ofhippocampus (Figures 1A and 1C) and cerebellum (Figures 1B and 1D). Many immunoreactive spots of anti-6-NO2Trp antibody were observed inboth of the brain regions from the 6-month-old adult rats. Although several immunoreactive spotswere detected only in hippocampus or cerebellum, most of the immunoreactive spots were observed inboth. In the control experiment, NO2Trp was reduced (Figures 2C and 2D) by the method described in the text.The results of the same treatment without reducing agents are shown in Figures 2(A) and 2(B). The immunoreactive spots ofrabbit polyclonal and mouse monoclonal anti-6-NO2Trp antibody became weak and severalspots disappeared by the reduction. Although complete disappearance of the spots had not beenattained by this treatment, it has been reported that reduction can be problematic and sometimes maynot fully reduce all of the nitrated protein present [21].Therefore these results indicate the specificity of both anti-6-NO2Trp antibodies isconsiderably high.


Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum.

Uda M, Kawasaki H, Shigenaga A, Baba T, Yamakura F - Biosci. Rep. (2012)

Representative results of Western blotting with an anti-6-NO2Trp antibody andSypro Ruby-stained gel after two-dimensional electrophoresis(A) and (C) are Western blotting and Sypro Ruby gel staining ofhippocampus respectively. (B) and (D) are those of cerebellum. Opencircled spots and spots indicated by arrows in hippocampus (h1–h16) and cerebellum(c1–c15) were subjected to LC-ESI-MS/MS analysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475453&req=5

Figure 1: Representative results of Western blotting with an anti-6-NO2Trp antibody andSypro Ruby-stained gel after two-dimensional electrophoresis(A) and (C) are Western blotting and Sypro Ruby gel staining ofhippocampus respectively. (B) and (D) are those of cerebellum. Opencircled spots and spots indicated by arrows in hippocampus (h1–h16) and cerebellum(c1–c15) were subjected to LC-ESI-MS/MS analysis.
Mentions: Figure 1 shows representative results of Western blottingwith the anti-6-NO2Trp antibody (Figures 1A and1B) and Sypro Ruby staining gels (Figures 1C and 1D) after two-dimensional PAGE ofhippocampus (Figures 1A and 1C) and cerebellum (Figures 1B and 1D). Many immunoreactive spots of anti-6-NO2Trp antibody were observed inboth of the brain regions from the 6-month-old adult rats. Although several immunoreactive spotswere detected only in hippocampus or cerebellum, most of the immunoreactive spots were observed inboth. In the control experiment, NO2Trp was reduced (Figures 2C and 2D) by the method described in the text.The results of the same treatment without reducing agents are shown in Figures 2(A) and 2(B). The immunoreactive spots ofrabbit polyclonal and mouse monoclonal anti-6-NO2Trp antibody became weak and severalspots disappeared by the reduction. Although complete disappearance of the spots had not beenattained by this treatment, it has been reported that reduction can be problematic and sometimes maynot fully reduce all of the nitrated protein present [21].Therefore these results indicate the specificity of both anti-6-NO2Trp antibodies isconsiderably high.

Bottom Line: We also observed that the total amount of NO2Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05).The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus.This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.

View Article: PubMed Central - PubMed

Affiliation: Sportology Center, Juntendo University Graduate School of Medicine, Chiba, Japan.

ABSTRACT
Nitration of tryptophan residues is a novel post-translational modification. In the present study, we examined whether NO2Trp (nitrotryptophan)-containing proteins are produced in the hippocampus and cerebellum of the adult rat under physiological conditions in vivo. Using Western blot analysis with anti-6-NO2Trp-specific antibody, we found many similar immunoreactive spots in the protein extracts from both regions. These spots were subsequently subjected to trypsin digestion and LC-ESI-MS/MS (LC-electrospray ionization-tandem MS) analysis. We identified several cytoskeletal proteins and glycolytic enzymes as NO2Trp-containing proteins and determined the position of nitrated tryptophan residues with significant ion score levels (P<0.05) in several proteins in both regions. We also observed that the total amount of NO2Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05). Moreover, IP (immunoprecipitation) assays using anti-aldolase C antibody showed that the relative intensity of immunostaining for NO2Trp over aldolase C was much higher in cerebellum than in hippocampus. The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus. This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.

Show MeSH
Related in: MedlinePlus