Oxidative stress is thought to be implicated in a number of age-associated disorders and neurodegenerative diseases. Dopamine neurons are known to be vulnerable to age-related disorders because of exposure to high level of oxidative stress by dopamine metabolism in neurons. Previously, we found that 6-hydroxydopamine-induced (6-OHDA, a dopaminergic neurotoxin) oxidative stress made changes in phosphorylation of nuclear lamin A/C, elongation factor 2, T-complex protein 1, and heterogeneous nuclear ribonucleoprotein H3 using human neuroblastoma cell line SH-SY5Y cells.
To understand the molecular basis of cellular response to oxidative stress, detailed information of protein-protein interaction under physiological (native) conditions is required. Here we further examined oxidative stress-related changes of SH-SY5Y cellular proteins by using blue-native polyacrylamide gel electrophoresis (BN-PAGE), a powerful tool for the separation of protein complexes. BN-PAGE gel images showed successful separation of several complexes. Components of these complexes, separated by 2-D BN-PAGE in combination with SDS-PAGE, were identified by peptide mass fingerprinting on MALDI-TOF MS and MS/MS ion search on LC-MS/MS. TCP-1 complex, ATP synthase, and the complex of heat shock protein 90 and its client proteins such as pyruvate kinase were detected. Several proteins were shown to be changed by oxidative stress. Identification of these proteins is now in progress.