Diagonal electrophoresis is a specialized technique used in biochemistry to separate and identify proteins based on the presence of disulfide bonds. Disulfide bonds are covalent links formed between the R groups of two cysteine residues, which can occur within the same polypeptide chain or between different chains. This method allows researchers to determine the positions of these bonds, which is crucial for understanding protein structure and function.
In diagonal electrophoresis, proteins are first fragmented, typically using enzymes like peptidases or chemical agents that cleave the protein into smaller pieces. Following fragmentation, the protein fragments are separated using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), which sorts proteins based on their molecular size. Notably, fragments that are disulfide-linked will migrate together and appear as a single band on the gel, while those without disulfide bonds will align along a diagonal line due to their unchanged mobility.
After the initial separation, the gel is exposed to performic acid vapors, which cleave the disulfide bonds. This step is critical as it allows the previously linked fragments to separate based on their actual sizes. The gel is then rotated, and a second SDS-PAGE run is performed in a perpendicular direction. This results in a diagonal line on the gel, where fragments without disulfide bonds align along the diagonal, while those with disulfide bonds appear off the diagonal due to their altered migration patterns.
The final step involves isolating the disulfide-linked peptides from the gel for sequencing. This sequencing reveals the specific positions of the disulfide bonds, providing valuable insights into the protein's structure. For example, a biochemist may determine that cysteine residues at positions 36 and 54 are disulfide bonded, while cysteine at position 72 is not. Thus, diagonal electrophoresis serves as an effective tool for identifying disulfide-linked proteins and understanding their structural relationships.