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Simultaneous detection of changes in protein expression and oxidative modification as function of age and APOE genotype in human APOE-targeted replacement mice
DeKroon, R., C. Osorio, J. Robinette, M. Mocanu, W. M. WINNIK, AND O. Alzate. Simultaneous detection of changes in protein expression and oxidative modification as function of age and APOE genotype in human APOE-targeted replacement mice. Journal of Proteome Research . American Chemical Society, Washington, DC, 10(4):1632-44, (2011).
Background The purpose of this study was to improve the current method for detecting differentially-oxidized (carbonyl-modified) proteins by 2D-DIGE, while at the same time determining changes in total protein expression. Protein oxidation is a widely accepted model of aging and Alzheimer's disease. To study changes in the level of oxidation we used hippocampi from young (25-30 weeks) and old (76-97 weeks) mice transgenic for the human Apolipoprotein E gene isoforms, APOE3 or APOE4. APOE4 being a risk factor for Alzheimer's disease. Samples were labelled with either a fluorescent aminooxyacetamide (Alexa Fluor 488) to detect carbonyl modifications, or NHS-Cy3 to detect total protein expression. An internal control was labelled with NHS-Cy5 and run on each gel. Results An apparent shift in pI was seen with the aminooxyacetamide label of oxidized proteins compared to Cy3 amine-labelled proteins. DIGE analysis revealed 38 oxidized and 100 total protein spots with significantly different (P<0.05) expression levels. For oxidized proteins, principal component analysis revealed two distinct clusters:one in which oxidation increased with age independent ofAPOE-genotype, and the second where oxidation was dependent on APOE genotype.For total protein expression, principal component analysis revealed a large overlap between overall aging changes and those between APOE genotypes. Conclusions In conclusion, the use ofa fluorescent label against oxidized proteins in combination with a NHS-Cy3 label for total protein makes it possible to determine differences in protein oxidation and total protein expression in a single study.
Describes new method of oxidized protein 2D gel separation, quantitation and ID in mice brain (young vs old and transgenic). It will benefit NHEERL studies by U. Kodavanti: Mitochondrial oxidative stress and cardiovascular. IRP-NHEERL-RTP/ETD/PTB/U.Kodavanti/05/00-r1
Record Details:Record Type: DOCUMENT (JOURNAL/PEER REVIEWED JOURNAL)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH LAB
ASSOCIATE DIRECTOR FOR HEALTH
RESEARCH CORES UNIT