Proteomic analysis of protein oxidation in Alzheimer's disease brain.
There is a growing body of evidence that oxidative stress plays a major role in Alzheimer's disease (AD) pathogenesis. Identification of oxidatively altered proteins in AD is important for understanding the relationship between protein oxidation, protein aggregation and neurodegeneration. In this communication, we report a method that can be applied to study oxidative changes of individual proteins in brain. In order to analyze protein oxidation by detection of protein-bound carbonyls, cytosolic protein extracts were derivatized with 2,4-dinitrophenylhydrazine (DNPH) and then separated by two-dimensional (2-D) gel electrophoresis. After electrotransfer to polyvinylidene difluoride (PVDF) membranes, proteins were first stained with Sypro Ruby protein stain, and then the oxidized proteins were detected with anti-dinitrophenyl (DNP) antibody. About 150 proteins and more than 100 oxidized proteins were detected and quantified in both AD and control cases by 2-D image analysis. The amount of protein-bound carbonyls was decreased for six and increased for one protein in AD. The amount of protein was increased for three proteins in AD. Furthermore, the degree of oxidation was calculated as the ratio of protein-bound carbonyls to the total amount of an individual protein. Two proteins showed a significant decrease in the degree of oxidation in AD. Our results suggest that the balance of protein oxidation and degradation is altered in AD.
Korolainen
Minna A
MA
Department of Neuroscience and Neurology, A.I Virtanen Institute for Molecular Sciences, University of Kuopio, Finland. minna.korolainen@uku.fi
Goldsteins
Gundars
G
Alafuzoff
Irina
I
Koistinaho
Jari
J
Pirttilä
Tuula
T
D000368:Aged; D000369:Aged, 80 and over; D000544:Alzheimer Disease / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007525:Isoelectric Focusing; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*
D011506:Proteins