In-gel Digestion - Reduction and Alkylation (r&a)

Reduction and Alkylation (r&a)

The staining and destaining is often followed by the reduction and alkylation (r&a) of the cystines or cysteines potentially embodied in the protein. Hereby the disulfide bonds of the proteins are irreversibly broken up and the optimal unfolding of the tertiary structure is obtained. The reduction to the thiol is accomplished by the reaction with chemicals containing sulfhydryl or phosphine groups such as dithiothreitol (DTT) or tris-2-carboxyethylphosphine hydrochloride (TCEP). In course of the subsequent irreversible alkylation of the SH groups with iodoacetamide the cysteines are transformed to the stable S-carboxyamidomethylcysteine (CAM; adduct: -CH₂-CONH₂). The specific mass of the aminoacid cysteine is thereby increased from 103.01 Da to 160.03 Da.

This chemical modification allows for proteins with a high number of disulfide bonds the successful identification as well as the highest peptide yield and sequence coverage. Due to the rareness of the aminoacid cysteine for most of the proteins the step of r&a does not effect any improvement of the mass spectrometric analysis. For the quantitative and homogeneous alkylation of cysteines the point of time for the modification is crucial. With denaturing electrophoresis it is strongly recommended to perform the reaction before the execution of the electrophoresis, since there are free acrylamide monomers in the gel able to modify cysteines. The resulting acrylamide adducts are bound irreversible to the cysteines and can not be removed by subsequent r&a. The specific mass of the adduct is 174.05 Da.