Determination of disulfide-bond linkages in proteins

The formation of disulfide bonds in proteins is an important post-translational modification that is critical for stabilizing the native structures of proteins. The disulfide linkages in a protein cannot be predicted from its amino acid sequence; therefore, determination of disulfide bond linkages in the protein will provide insights into its three-dimensional structure and contribute to the understanding of its structural-functional relationship. This unit details a series of protocols that have been applied successfully to locate disulfide bonds in proteins. The general strategy involves chemical or proteolytic cleavage of the protein followed by chromatographic separation of the resultant peptides. Disulfide-containing peptides are identified by the alteration of mobility as a consequence of disulfide bond reduction, and are further characterized by mass spectrometry and/or N-terminal sequencing. A partial reduction and alkylation strategy for mapping disulfide linkages in peptides with multiple disulfide bonds is also presented.