Journal
EXPERIMENTAL GERONTOLOGY
Volume 44, Issue 6-7, Pages 375-382Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.exger.2009.02.009
Keywords
Lamin A/C; Oxidative stress; SH-SY5Y cell; 6-Hydroxy dopamine; Blue-native electrophoresis; Proteomics
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Funding
- Japan Society for the Promotion of Science
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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. (C) 2009 Elsevier Inc. All rights reserved.
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