Disulfide linkage assignment based on reducing electrochemistry and mass spectrometry using a lead electrode

The study of disulfide linkage is a crucial part of the quality assessment of biopharmaceutical products because disulfide bonds stabilize the tertiary structure of proteins and maintain protein functions. Therefore, a suitable method is highly required for disulfide linkage assignment when nested disulfide bonds formed with closely spaced cysteine residues. A novel approach for disulfide linkage assignment of disulfide-rich peptides and proteins via electrochemical reduction on a lead electrode with mass spectrometry is presented in this paper. The method features partial electrochemical reduction and alkylation of peptides followed by alkylated peptide sequencing based on tandem mass spectrometry. Lead was chosen for the first time as an electrode material for disulfide bond reduction, because it has the advantages of maintenance free (only infrequent polishing needed), easy operation in DC mode, and reusability. Without any special sample preparation and any chemical reduction agents, disulfide bond in peptides can be cleaved rapidly. The new method was successfully tested with two peptides and one protein containing nested disulfide bonds.