Simultaneous detection of thiol- and disulfide-containing peptides by electrochemical high-performance liquid chromatography with identification by mass spectrometry

A new HPLC electrochemical detector that can be used to detect selectively and simultaneously both thiol- and disulfide-containing peptides is described. One electrode responds only to thiol-containing peptides, while a second electrode located downstream from a third electrode responds to thiol- as well as disulfide-containing peptides. All three electrodes are located in a single block and sample the HPLC effluent simultaneously. Since the electrochemical detector responds only to peptides that contain thiol or disulfide functionalities, it is ideal for detecting these types of peptides when they are present in complex mixtures. Peptides can be identified by their retention times if reference peptides are available, or by their molecular weights if the appropriate chromatographic fractions are collected and analyzed by mass spectrometry. The utility of the three-electrode electrochemical detector for detecting thiol- and disulfide-containing peptides in a single chromatographic analysis is illustrated with proteolytic digests of bovine alpha A-crystallin and partially reduced bovine insulin.