Abstract
The formation of disulfide bonds in proteins is an important post-translational modification that is critical for stabilizing the native structures of proteins, particularly proteins exposed to oxidizing environments. For this reason, most cysteines in secreted proteins or protein domains on the surface of the cell are in disulfides, whereas most cysteines in the cytoplasm are in the unmodified -SH form. Disulfide linkages must be experimentally determined, as they cannot be predicted from amino acid sequence. These assignments provide insights into three-dimensional structure and contribute to the understanding of structural-functional relationships. This unit details a series of protocols that have been applied successfully to map disulfide bonds in proteins. The general strategy involves chemical or proteolytic cleavage of the protein followed by chromatographic separation of the resultant peptides. Mass spectrometry is used to identify disulfide-containing peptides and determine sites of disulfide linkage. A partial reduction and alkylation strategy for mapping disulfide linkages in peptides with multiple disulfide bonds is also presented. © 2019 by John Wiley & Sons, Inc.