Novel approach to rapid and sensitive localization of protein disulfide bridges by high-performance liquid chromatography and electrochemical detection

• Published in: Journal of Chromatography A 1985-01, Vol.326, p.339-348
• Main Authors: Lazure, Claude, Rochemont, Jim, Seidah, Nabil G., Chrétien, Michel
• Format: Article
• Language: English
• Subjects: chromatography; disulfide bonds; high-performance liquid chromatography; U.V. spectroscopy
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(01)87459-8

No method has yet been developed for the chemical localization of a disulfide linkage which is capable of using the very low amounts of proteins or polypeptides often encountered today. The novel approach reported herein is entirely based on the use of high-performance liquid chromatography; hence it offers the rapidly, reliability and sensitivity necessary in order to solve this most important problem. The first step involves the separation of a proteolytic digest of the native protein by reversed-phase chromatography. The separation is monitored using a UV spectrophotometer coupled to an electrochemical detector able to detect solely disulfide-containing peptides. The second step involves the chemical reduction and alkylation of these peptides followed by purification under the same conditions as the initial run. Amino acid composition allows identification of each peptide and its precise localization in the protein sequence. This report is meant to demonstrate the utility of the electrochemical detector for detecting disulfide-containing peptides and to validate the feasibility of this proposed approach, byusing as a model a 26-amino acid peptide atrial natriuretic factor containing one disulfide bridge.