Identification of protein carbonyls after two-dimensional electrophoresis

Abstract

The oxidative modification of proteins plays a major role in a number of human diseases, but identity of the specific proteins that are most susceptible to oxidation has posed a difficult problem. Protein carbonyls are increased after oxidative stress, and after derivatization with 2,4-dinitrophenyl hydrazine (DNP) they can be detected by various analytical and immunological methods. Although high resolution two-dimensional electrophoresis (2-DE) can resolve virtually all proteins present in a cell or tissue it has been difficult to determine the oxidized proteins because the DNP-derivatization process alters the isoelectric points of proteins, and additional procedures must be utilized to remove reaction byproducts. These additional procedures can lead to loss of sample, and poor isoelectric resolution on immobilized pH gradient (IPG) strips. We have developed a method that allows the IPG strips to be derivatized with DNP directly following isoelectric focusing of the proteins. This method allows the visualization of oxidized proteins by 2-DE with high reproducibility.

Oxidized proteins in plasma of patients with heart failure: Role in endothelial damage

“…300 μg of plasma proteins, measured by Bradford method, were then diluted with a buffer yielding final concentrations of 7 mol/L urea, 2 mol/L thiourea, 0.2% SDS, 4% CHAPS, 2% v/v carrier ampholytes pH 3–10, 20 mmol/L Tris, 55 mmol/L dithiothreitol, and bromophenol blue. IPG ready strips, 11 cm, pH 3–10 non linear gradient or 4–7 linear gradient (Biorad), were actively rehydrated at 50 V for 24 h. Samples were loaded at the cathode and focused for a total of 20 kV h. Following isoelectric focusing, the oxidized proteins were derivatized by the “in‐strip DNPH derivatization” method of Conrad et al [9]. Briefly, the IPG strips were incubated in 2 N HCl with 10 mmol/L DNPH, washed with 2 mol/L Tris/30% glycerol and equilibrated first with a solution containing 50 mmol/L Tris‐HCl, 6 mol/L urea, 30% v/v glycerol, 2% SDS and 2% dithiothreitol, and then with the same buffer containing 4.5% iodoacetamide instead of dithiothreitol.…”

“…Before immunodetection, the membranes were stained in 0.2% w/v Ponceau S in 3% w/v trichloroacetic acid and the spot position marked in order to facilitate computer‐assisted matching on the colloidal blue stained gel. Immunodetection was performed as previously described [9], using biotinylated anti‐DNP antibody (Molecular Probes). The immunoreactive spots were detected using avidin horseradish peroxidase‐conjugated (Biorad) and a chemiluminescence detection system (Amersham).…”

Copper‐induced oxidative stress in Saccharomyces cerevisiae targets enzymes of the glycolytic pathway

“…The general methods for 2D protein analysis and Western blotting are adapted from Ausubel et al [25] with modifications for immunodetection of protein carbonyl groups according to [20,26]. For 2D analysis either 40 μg (for protein detection) or 100 μg (for Western blotting) of protein was mixed with re‐hydration solution (8 M urea, 0.4% dithiothreitol (DTT), 4% CHAPS and 1% immobilized pH gradient (IPG) buffer, pH 3–10) and loaded on to 18 cm IPG strips (non‐linear pH gradient 3–10; Amersham Pharmacia).…”

“…The membranes were incubated in phosphate‐buffered saline–Tween containing 5% (w/v) skimmed milk powder, and were probed with rabbit anti‐DNP as primary antibody (Molecular Probes Inc; 1:16 000 dilution) and peroxidase‐linked goat‐anti rabbit IgG as secondary antibody (Sigma; 1:16 000 dilution). Carbonylated proteins were immunodetected with a chemiluminescent peroxidase substrate, West femtoM (Pierce) [26] and visualized using a Fuji LAS1000 image analyzer with pre‐cooled camera. Chemiluminescence was quantified using Fuji image gauge software.…”

Identification and characterization of molecular targets of natural products by mass spectrometry

“…England, Driscoll, and Cotter (2006) analyzed the redox proteomics of HL‐60 leukemia cells treated with doxorubicin, etoposide, and mitoxantrone. The strategy involved separation of a whole‐cell proteome by isoelectric focusing, after which the gel strip was stained with DNPH before the second dimension SDS–PAGE (Conrad et al, 2001). This strategy minimized problems associated with DNPH‐induced alterations of the isoelectric point of proteins and DNPH‐derived contaminants.…”