Diagonal Electrophoresis for Detection of Protein Disulphide Bridges

A state of oxidative stress (OS) can occur when there is an imbalance between the rate of reactive oxygen species (ROS) production and their detoxification. Under OS conditions sulphur-containing residues are particularly susceptible to oxidation, and this can result in transient formation of intra- or inter-chain disulphide bridges. Diagonal electrophoresis is a relatively simple technique to analyse the formation of these bridges by sequential non-reducing/reducing electrophoresis. Proteins that do not form disulphides, electrophorese identically in both dimensions and form a diagonal after the second dimension, proteins that contained intra-chain disulphides lie above this diagonal, while those that formed inter-disulphides fall below the diagonal. These spots can be excised, tryptic digested, and identified by mass spectrometry. Identification of those proteins, which are reversibly modified, could play an important part in coupling redox status to protein function.