Limits...
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.

Show MeSH

Related in: MedlinePlus

MS/MS spectrum of the tryptic peptide 304R.ALQASALSA314W-NO2R. G315 from fructose-bisphosphate aldolaseCW-NO2 indicates NO2Trp residue.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3475453&req=5

Figure 3: MS/MS spectrum of the tryptic peptide 304R.ALQASALSA314W-NO2R. G315 from fructose-bisphosphate aldolaseCW-NO2 indicates NO2Trp residue.

Mentions: A total of 16 immunoreactive spots and 15 spots on the membrane for the samples from hippocampusand cerebellum respectively were cut out from Sypro Ruby-stained gels and digested with trypsin. Thedigested samples were analysed with LC-ESI-MS/MS. Putative 6-NO2Trp-containing proteinsfrom both regions are shown in Table 1. Although fourproteins including mitochondrial electron transfer flavoprotein subunit α and creatinekinases were identified only in hippocampus and three proteins including voltage-dependentanion-selective channel protein 1 were identified only in cerebellum as putative6-NO2Trp-containing proteins (Table 1), most ofthe other spots included the same 12 proteins, such as α-enolase, tubulins andfructose-bisphosphate aldolases (Table 1). We successfullydetermined the positions of the nitrated tryptophan residues in amino acid sequences of 12 proteinsfrom hippocampus (Table 2) and seven proteins from cerebellum(Table 3). These proteins in both regions includedcytoskeletal proteins and glycolytic enzymes. Figure 3 showsthe MS/MS spectrum of fructose-bisphosphate aldolase C as a typical example of the MS/MSanalyses.


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)

MS/MS spectrum of the tryptic peptide 304R.ALQASALSA314W-NO2R. G315 from fructose-bisphosphate aldolaseCW-NO2 indicates NO2Trp residue.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: MS/MS spectrum of the tryptic peptide 304R.ALQASALSA314W-NO2R. G315 from fructose-bisphosphate aldolaseCW-NO2 indicates NO2Trp residue.
Mentions: A total of 16 immunoreactive spots and 15 spots on the membrane for the samples from hippocampusand cerebellum respectively were cut out from Sypro Ruby-stained gels and digested with trypsin. Thedigested samples were analysed with LC-ESI-MS/MS. Putative 6-NO2Trp-containing proteinsfrom both regions are shown in Table 1. Although fourproteins including mitochondrial electron transfer flavoprotein subunit α and creatinekinases were identified only in hippocampus and three proteins including voltage-dependentanion-selective channel protein 1 were identified only in cerebellum as putative6-NO2Trp-containing proteins (Table 1), most ofthe other spots included the same 12 proteins, such as α-enolase, tubulins andfructose-bisphosphate aldolases (Table 1). We successfullydetermined the positions of the nitrated tryptophan residues in amino acid sequences of 12 proteinsfrom hippocampus (Table 2) and seven proteins from cerebellum(Table 3). These proteins in both regions includedcytoskeletal proteins and glycolytic enzymes. Figure 3 showsthe MS/MS spectrum of fructose-bisphosphate aldolase C as a typical example of the MS/MSanalyses.

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