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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
Citation:
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).
Impact/Purpose:
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
Description:
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.
