Oxidative stress is thought to be implicated in a number of age-associated disorders, neurodegenerative diseases, and aging mechanism itself. Dopamine neurons are known to be vulnerable to age-related disorders because of exposure to high level of oxidative stress by dopamine metabolism in these neurons. Here we examined the overall protein alterations of human neuroblastoma cell line SH-SY5Y under oxidative stress induced by 6-hydroxydopamine (6-OHDA), a dopaminergic neurotoxin. After exposure to 6-OHDA, cell injury was evaluated by a colorimetric assay for mitochondrial function using the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate (WST-1) test and cellular proteins were analyzed by two-dimensional gel-electrophoresis in combination with MALDI-TOF mass spectrometry. The phosphorylation state of entire proteomes was analysed by using Pro-Q Diamond dye. The levels of free ubiquitin and ubiquitin-conjugated proteins were examined by Western blot analysis using anti-ubiquitin antibody. Differential display of proteins using computer-aided image analysis revealed that several protein spots were shown to be varied both in quantity and in quality as the effect of oxidative stress. Identification of these protein spots and their oxidative stress-related modification is now in progress.
