Phosphoproteomic profiling of human SH-SY5Y neuroblastoma cells during response to 6-hydroxydopamine-induced oxidative stress.
Dopaminergic neurons are known to be vulnerable to age-related neuronal disorders due to reactive oxygen species (ROS) generated during dopamine metabolism. However, it remains unclear what kinds of proteins are involved in the response to oxidative stress. We examined changes in whole proteins and phosphoproteins in the human dopaminergic neuroblastoma cell line SH-SY5Y under oxidative stress induced by the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Proteins of SH-SY5Y cells at various stages of oxidative stress were separated by two-dimensional gel electrophoresis for comparative analysis. Increase in glutathione-S-transferase pi was detected on SYPRO Ruby-stained gels by computer-aided image analysis. Stress-induced alterations in phosphoproteins were detected by Pro-Q Diamond staining. Elongation factor 2, lamin A/C, T-complex protein 1, and heterogeneous nuclear ribonucleoprotein H3 were identified by MALDI-TOF mass spectrometry as stress-responsive elements.
Nakamura
Megumi
M
Research Team for Molecular Biomarkers, Proteomic Gerontology Research Unit, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan.
Yamada
Masaki
M
Ohsawa
Takako
T
Morisawa
Hiraku
H
Nishine
Tsutomu
T
Nishimura
Osamu
O
Toda
Tosifusa
T
D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D015180:Electrophoresis, Gel, Two-Dimensional; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D018384:Oxidative Stress / Q000187:drug effects*; D016627:Oxidopamine / Q000633:toxicity*; D010750:Phosphoproteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010750:Phosphoproteins; D016627:Oxidopamine