Redox proteomic analysis of carbonylated brain proteins in mild cognitive impairment and early Alzheimer's disease

Abstract

Previous studies indicated increased levels of protein oxidation in brain from subjects with Alzheimer's disease (AD), raising the question of whether oxidative damage is a late effect of neurodegeneration or precedes and contributes to the pathogenesis of AD. Hence, in the present study we used a parallel proteomic approach to identify oxidatively modified proteins in inferior parietal lobule (IPL) from subjects with mild cognitive impairment (MCI) and early stage-AD (EAD). By comparing to age-matched controls, we reasoned that such analysis could help in understanding potential mechanisms involved in upstream processes in AD pathogenesis. We have identified four proteins that showed elevated levels of protein carbonyls: carbonic anhydrase II (CA II), heat shock protein 70 (Hsp70), mitogen-activated protein kinase I (MAPKI), and syntaxin binding protein I (SBP1) in MCI IPL. In EAD IPL we identified three proteins: phosphoglycerate mutase 1 (PM1), glial fibrillary acidic protein, and fructose bisphospate aldolase C (FBA-C). Our results imply that some of the common targets of protein carbonylation correlated with AD neuropathology and suggest a possible involvement of protein modifications in the AD progression.

FIG. 1.

Representative SYPRO Ruby 2-D gels of the inferior parietal lobule from control (A) or MCI (B) brain. Protein (150 μg) was separated on immobilized pH 3–10 IPG strips, followed by separation on an 8–16% gradient SDS-PAGE gels.

FIG. 2.

Two-dimensional carbonyl immunoblots from MCI and control (CTR) subjects (IPL). Positions of the four identified protein are shown on the blots. Expanded images of the selected proteins on the blots are also shown. Relative change in carbonyl immunoreactivity, after normalization of the immunostaining intensities to the protein content, was significant for four proteins. See text. Expanded images of the selected proteins on the blots are also shown.

FIG. 3.

Two-dimensional carbonyl immunoblots from EAD and CTR subjects (IPL). Positions of the three identified proteins are shown on the blots.

FIG. 4.

Two-dimensional carbonyl immunoblots of CTR (A) and MCI (B) samples after immunoprecipitation with specific anti-Hsp70 antibody. To confirm the correct identification of Hsp70 by mass spectrometry, control or MCI samples (150 μg) were immunoprecipitated with anti-Hsp70 antibody and protein resolved by SDS-PAGE, followed by immunoblotting on nitrocellulose.

FIG. 5.

(A, left) 2D electrophoresis gel from supernatant of MCI IPL homogenate described in this study after immunoprecipitation by anti-CA II antibody. (B, right) 2D electrophoresis gel from the same sample without immunoprecipitation. The arrowhead indicates that CA II disappears after immunoprecipitation, confirming its identity.