Nitrotyrosine formation is caused by presence of reactive oxygen and nitrogen species. Nitration is a very selective process leading to specific modification of only a few tyrosines in protein molecule. 2D electrophoresis and western blotting techniques coupled with mass spectrometry are common methods used in analysis of proteome. Here we describe protocol for analysis of peroxynitrite-induced protein nitration in isolated mitochondria. Mitochondrial proteins are separated by 2D electrophoresis and transferred to nitrocellulose membrane. Membranes are then incubated with antibodies against nitrotyrosine. Positive spots are compared with corresponding Coomassie-stained gels, and protein nitration is confirmed with mass spectrometry techniques.

• Klíčová slova
• 2D electrophoresis, Immunoblotting, Mass spectrometry, Mitochondria, Nitrotyrosine,
• MeSH
• 2D gelová elektroforéza metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• imunoblotting metody   MeSH
• kyselina peroxydusitá chemie   MeSH
• mitochondriální proteiny analýza   metabolismus   MeSH
• skot MeSH
• srdeční mitochondrie chemie   metabolismus   MeSH
• tyrosin analogy a deriváty   analýza   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3-nitrotyrosine MeSH Prohlížeč
• kyselina peroxydusitá MeSH
• mitochondriální proteiny MeSH
• tyrosin MeSH

Oxidative stress after birth led us to localize reactive oxygen and nitrogen species (RONS) production in the developing rat brain. Brains were assessed a day prenatally and on postnatal days 1, 2, 4, 8, 14, 30, and 60. Oxidation of dihydroethidium detected superoxide; 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate revealed hydrogen peroxide; immunohistochemical proof of nitrotyrosine and carboxyethyllysine detected peroxynitrite formation and lipid peroxidation, respectively. Blue autofluorescence detected protein oxidation. The foetuses showed moderate RONS production, which changed cyclically during further development. The periods and sites of peak production of individual RONS differed, suggesting independent generation. On day 1, neuronal/glial RONS production decreased indicating that increased oxygen concentration after birth did not cause oxidative stress. Dramatic changes in the amount and the sites of RONS production occurred on day 4. Nitrotyrosine detection reached its maximum. Day 14 represented other vast alterations in RONS generation. Superoxide production in arachnoidal membrane reached its peak. From this day on, the internal elastic laminae of blood vessels revealed the blue autofluorescence. The adult animals produced moderate levels of superoxide; all other markers reached their minimum. There was a strong correlation between detection of nitrotyrosine and carboxyethyllysine probably caused by lipid peroxidation initiated with RONS.

• MeSH
• fluorescenční mikroskopie MeSH
• glykosylace MeSH
• kyselina peroxydusitá metabolismus   MeSH
• lysin analogy a deriváty   metabolismus   MeSH
• mozek růst a vývoj   metabolismus   MeSH
• novorozená zvířata MeSH
• oxidační stres * MeSH
• peroxid vodíku metabolismus   MeSH
• peroxidace lipidů MeSH
• posttranslační úpravy proteinů MeSH
• potkani Wistar MeSH
• reaktivní formy dusíku metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• superoxidy metabolismus   MeSH
• tyrosin analogy a deriváty   metabolismus   MeSH
• věkové faktory MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 3-nitrotyrosine MeSH Prohlížeč
• kyselina peroxydusitá MeSH
• lysin MeSH
• N(6)-carboxyethyllysine MeSH Prohlížeč
• peroxid vodíku MeSH
• reaktivní formy dusíku MeSH
• reaktivní formy kyslíku MeSH
• superoxidy MeSH
• tyrosin MeSH