Proteomic analysis of protein complexes in human SH-SY5Y neuroblastoma cells by using blue-native gel electrophoresis: an increase in lamin A/C associated with heat shock protein 90 in response to 6-hydroxydopamine-induced oxidative stress.
There is accumulating evidence that oxidative stress plays an important role in aging. Our previous phosphoproteomic study of the human neuroblastoma cell line SH-SY5Y revealed changes in the phosphorylation of several proteins such as lamin A/C during 6-hydroxydopamine-induced oxidative stress. The present study employed native proteomic analysis to clarify protein-protein interaction under physiological conditions. We examined oxidative stress-related changes in SH-SY5Y cellular proteins 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 employing MALDI-TOF MS and an MS/MS ion search on LC-MS/MS. TCP-1 complex, ATP synthase, and the complex of heat shock protein 90 with its client proteins such as pyruvate kinase were detected. Two dimensional BN-PAGE and Western blot analysis revealed an increase of lamin A/C associated with heat shock protein 90 in response to 6-OHDA-induced oxidative stress.
Nakamura
Megumi
M
Research Team for Molecular Biomarkers, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0015, Japan.
Morisawa
Hiraku
H
Imajoh-Ohmi
Shinobu
S
Takamura
Chizuko
C
Fukuda
Hiroyuki
H
Toda
Tosifusa
T
D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D030562:Databases, Protein; D042967:Electron Transport Complex I / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005782:Gels; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D006892:Hydroxydopamines / Q000378:metabolism*; D034904:Lamin Type A / Q000378:metabolism*; D013058:Mass Spectrometry; D009447:Neuroblastoma / Q000235:genetics*; D018384:Oxidative Stress / Q000235:genetics*
D005782:Gels; D018841:HSP90 Heat-Shock Proteins; D006892:Hydroxydopamines; D034904:Lamin Type A; D042967:Electron Transport Complex I