Detection of disulfide linkage by chemical derivatization and mass spectrometry.

The location of disulfide linkage(s) or status of unpaired cysteines is a critical structural feature required for the characterization of three-dimensional structure of a protein and for the correlation of protein structure-function relationships. Cysteine, with its reactive thiol group, can undergo enzymatic or oxidative posttranslational modification in response to changing redox conditions to signal a cascade of downstream reactions. In such a situation, it becomes even more critical to obtain the information on the pair of cysteines involved in such a redox switch operation. Here, a method involving chemical derivatization and liquid chromatography-mass spectrometry (LC-MS) is described to determine the cysteine residues involved in disulfide bond formation for a protein containing multiple cysteines in its sequence.