title: Simultaneous detection of changes in protein expression and oxidative modification as a function of age and APOE genotype.
abstract: 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.
authors.affiliation: UNC Systems-Proteomics Center, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, United States.
authors.forename: Robert M
authors.initials: RM
authors.lastname: DeKroon
authors.forename: Cristina
authors.initials: C
authors.lastname: Osorio
authors.forename: Jennifer B
authors.initials: JB
authors.lastname: Robinette
authors.forename: Mihaela
authors.initials: M
authors.lastname: Mocanu
authors.forename: Witold M
authors.initials: WM
authors.lastname: Winnik
authors.forename: Oscar
authors.initials: O
authors.lastname: Alzate
mesh_terms: D000375:Aging / Q000502:physiology*; D000818:Animals; D053318:Apolipoprotein E3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053327:Apolipoprotein E4 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005838:Genotype; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008822:Mice, Transgenic; D010084:Oxidation-Reduction; D025341:Principal Component Analysis; D057825:Two-Dimensional Difference Gel Electrophoresis / Q000379:methods
chemical_list: D053318:Apolipoprotein E3; D053327:Apolipoprotein E4