Post-Electrophoretic Identification of Oxidized Proteins

The oxidative modification of proteins has been shown to play a major role in a number of human diseases. However, the ability to identify specific proteins that are most susceptible to oxidative modifications is difficult. Separation of proteins using polyacrylamide gel electrophoresis (PAGE) offers the analytical potential for the recovery, amino acid sequencing, and identification of thousands of individual proteins from cells and tissues. We have developed a method to allow underivatized proteins to be electroblotted onto PVDF membranes before derivatization and staining. Since both the protein and oxidation proteins are quantifiable, the specific oxidation index of each protein can be determined. The optimal sequence and conditions for the staining process are (a) electrophoresis, (b) electroblotting onto PVDF membranes, (c) derivatization of carbonyls with 2,4-DNP, (d) immunostaining with anti DNP antibody, and (e) protein staining with colloidal gold

Anti-apoptotic proteins are oxidized by Aβ25–35 in Alzheimer's fibroblasts

AbstractWe have examined the effects of the beta-amyloid peptide (Aβ25–35) on fibroblasts derived from subjects with Alzheimer's disease (AD) and from age-matched controls. The peptide was significantly more cytotoxic to the AD-derived fibroblasts. The level of protein oxidation was also greater in the cells from AD subjects. Two-dimensional electrophoresis (2-DE) coupled with immunostaining for protein carbonylation revealed specific oxidation-sensitive proteins (OSPs) in both the control and AD-derived cells. Two specific OSPs were identified by mass spectrometry as heat shock protein 60 (HSP 60) and vimentin. Exposure of the cells to Aβ25–35 resulted in a twofold increase in the level of oxidation of these two OSPs in the cells derived from controls, but a ninefold increase in their level of oxidation in the fibroblasts from AD subjects. These observations are of particular interest because of the proposed anti-apoptotic roles of both HSP 60 and vimentin and our recent observation that these same two proteins are particularly susceptible to oxidation in neuronally derived cells