Diagonal Electrophoresis for the Detection of Proteins Involved in Disulfide Bonds

The formation and reduction of disulfide bonds through thiol-disulfide exchange is an important cellular mechanism for the regulation of protein activity and plays a crucial role in redox signaling. Thiol-disulfide exchange on key regulatory cysteine residues offers an efficient and reversible mechanism for regulatory control in response to alterations in the intracellular redox environment. Cysteines are one of the most rarely used amino acids in proteins, but when conserved in proteins, they usually play critical roles in protein structure, function, or regulation. Diagonal electrophoresis is a relatively simple technique to analyze the formation of protein disulfides by sequential nonreducing/reducing electrophoresis. Proteins that do not form disulfides migrate identically in both dimensions and form a diagonal after the second dimension; proteins that contained intra-chain disulfides lie above the diagonal, while proteins involved in inter-disulfides fall below the diagonal. This technique allows for the detection and subsequent identification of proteins with inter-disulfides and has been applied in combination with mass spectrometry to identify proteins involved in redox signaling cascades.