Multiplexed Proteomic Analysis of Oxidation and Concentrations of Cerebrospinal Fluid Proteins in Alzheimer Disease

Abstract

Background:Carbonylation is an irreversible oxidative modification of proteins that has been linked to various conditions of oxidative stress, aging, physiological disorders, and disease. Increased oxidative stress is thus also considered to play a role in the pathogenesis of age-related neurodegenerative disorders such as Alzheimer disease (AD). In addition, it has recently become evident that the response mechanisms to increased oxidative stress may depend on sex. Several oxidized carbonylated proteins have been identified in plasma and brain of AD patients by use of 2-dimensional oxyblotting. Methods: In this pilot study, we estimated the concentrations and carbonylation of the most abundant cerebrospinal fluid proteins in aging women and men, both AD patients suffering from mild dementia and individuals exhibiting no cognitive decline. Oxidized carbonylated proteins were analyzed with 2-dimensional multiplexed oxyblotting, mass spectrometry, and database searches. Results: Signals for ␤-trace, chain, and transthyretins were decreased in probable AD patients compared with controls. The only identified protein exhibiting an increased degree of carbonylation in AD patients was chain. The concentrations of proteins did not generally differ between men and women; however, vitamin

Age-related changes in the expression and oxidation of bronchoalveolar lavage proteins in the rat

“…To test this hypothesis, we used electrophoretic approaches, including 2D-DIGE (32,35), a powerful discovery proteomic technique, and another electrophoretic technique (29) we have modified that permits the relative carbonyl content of specific proteins to be assessed in specific proteins. Using 2D-DIGE, we resolve 583 protein spots in all of our gels.…”

“…Spots from the images of the membranes were also matched back into the 2D-DIGE experiment to get identities for some of the proteins with significant changes in oxidation. This method is similar to a previously published method (29), but several modifications have made to increase sensitivity. These include the replacement of Sypro Ruby with the more sensitive Deep Purple and the incorporation of a biotinylated second antibody and a Cy5-labeled streptavidin detection method that is several times more sensitive than detection with a Cy5-labeled secondary antibody alone (29).…”

“…This method is similar to a previously published method (29), but several modifications have made to increase sensitivity. These include the replacement of Sypro Ruby with the more sensitive Deep Purple and the incorporation of a biotinylated second antibody and a Cy5-labeled streptavidin detection method that is several times more sensitive than detection with a Cy5-labeled secondary antibody alone (29). Relative oxidation of each protein was determined by dividing the Cy5-OxyBlot volume by the Deep Purple volume for each protein.…”

“…Abbreviations for biological processes: AFRR, antioxidation and free radical removal; BAMI, B-cell-and antibodymediated immunity; CATT, cation transport; CC, cell cycle; CK, cytokinesis; CMI, complement-mediated immunity; CPGM, coenzyme and prosthetic group metabolism; CS, cell structure; DTX, detoxification; EE, exocytosis and endocytosis; ID, immunity and defense; LFSM, lipid, fatty acid and steroid metabolism; LMS, ligand-mediated signaling; MISC, miscellaneous; MMI, macrophage-mediated immunity; OBCGE, other blood circulation and gas exchange activity; OCP, other coenzyme and prosthetic group metabolism; OHA, other homeostasis activities; OID, other immune and defense; OPTL, other protein targeting and localization; PB, protein biosynthesis; PCXA, protein complex assembly; PF, protein folding; PROT, proteolysis; RME, receptor-mediated endocytosis; SD, skeletal development; SR, stress response; ST, signal transduction; TAP, tricarboxylic acid pathway; TRAN, transport; UN, biological process unclassified; VCT, vitamin/cofactor transport. approach has been described previously (29), but the modifications we have incorporated should markedly enhance its sensitivity. However, the Deep Purple stain was slightly less sensitive than the Cy dyes used for the 2D-DIGE studies, so fewer protein spots were detected.…”

Effect of Curcuma longa Hot Water Extract on Activity of Neuronal Cells Related to Oxidative Stress

“…신경퇴행성 장애로 인한 기억과 인지력이 상실되는 질병인 알츠하이머(Alzheimer's disease; AD)의 발생에 산화스트레 스가 중요한 역할을 하는 것으로 알려졌다 [16]. 최근에 AD에 서 핵심적 병리 소견의 하나인 신경섬유다발(neurofibrillary tangle)은 산화스트레스가 선행된 다음에 발생된다고 보고되 었다 [27].…”