KCS

A disulfide bond between cysteine residues, a post-translational modification, is the key elements of many secreted proteins and peptides. Recently, methods to isolate the disulfide bond, using a thiol affinity column, have been reported. In this work, we used gold clusters with free thiols attached to efficiently separate and detect the disulfide containing proteins. The gold clusters selectively form covalent bond with free thiol groups of proteins. We carried out separation of different forms of proteins that consist of either free thiols or disulfide bond or both. The fractions were analyzed by cRPLC/MS/MS. The result of LC/MS/MS experiment using the disulfide enriched samples demonstrated minimized contamination due to minimized non-specific binding of proteins, which commonly observed with conventional beads. The effectiveness of the disulfide capturing was compared with that of commercial thiol affinity column.