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Simultaneous Detection of Changes in Protein Expression and Oxidative Modification as a Function of Age and APOE Genotype

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posted on 01.04.2011, 00:00 authored by Robert M. DeKroon, Cristina Osorio, Jennifer B. Robinette, Mihaela Mocanu, Witold M. Winnik, Oscar Alzate
To better elucidate temporal changes in protein oxidation resulting from aging and the Alzheimer’s disease-associated Apolipoprotein E (ApoE), we developed a 2D-DIGE-based method for simultaneously detecting differential expression and carbonyl oxidation of proteins. Specifically, we examined changes in the levels of oxidation and total protein expression in hippocampi from young-adult (25−30 weeks) and old (76−97 weeks) mice transgenic for the human Apolipoprotein E gene (APOE, APOE3, APOE4) isoforms, APOE3 or APOE4. Protein samples were labeled with either a fluorescent aminooxyacetamide (Alexa Fluor 488) to detect carbonyl modifications or with NHS-Cy3 to detect total protein expression. A protein sample used as an internal control was labeled with NHS-Cy5 and run on each gel. DIGE analysis revealed 38 differentially oxidized and 100 differentially expressed protein spots with significantly different levels (P < 0.05). For oxidized proteins, principal component analysis revealed two distinct clusters: one in which oxidation increased with age independent of APOE genotype, and the second in which oxidation was dependent on APOE genotype. For total protein expression, principal component analysis revealed a large overlap between changes with overall aging and between APOE genotypes. The use of a fluorescent tag to label oxidized proteins, in combination with a NHS-Cy3 to label total protein, makes it possible to determine changes in both protein oxidation and protein expression levels in a single experiment. These studies reveal that the expression levels of peroxiredoxin protein family members Prdx2, 3, and 6 are modified by age, APOE genotype, or both.