Identification and characterisation of disulphide bonds in therapeutic proteins by using Raman Spectroscopy

Protein aggregation results in the formation of long, unbranched -sheet-rich structures, commonly known as amyloid fibrils, which are believed to be a defense mechanism against the small and most toxic aggregates of misfolded proteins. Disulfide bonds limit the way in which a protein or a peptide can aggregate into these fibrils via steric restraint. There is great interest in understanding the influence of disulfide bonds on the stability of therapeutic proteins such as insulin. Therefore, the identification and characterization of protein disulfide bonds is an essential step to thoroughly understand their biological function. However, the determination of disulfide linkages can be a challenging task. Several methods such as crystallography, NMR and mass spectrometry are used for the determination and identification of disulfide bond linkage; however, their application is limited by large sample requirements, protein size, ambiguous results and complexity of data analysis. Raman spectroscopy has several advantages over other spectroscopic techniques or chemical analyses e.g. ease of measurements, its versatility in application to aqueous solutions, nonaqueous liquids, fibers, films, powders, gels and crystals without destruction of the samples. Therefore, Raman spectroscopy was used in the present research to identify and obtain quantitative information of therapeutic proteins. Solid samples of cysteine and cystine were used to identify and quantitate the ratio of disulfide and S-H bonds. The information obtained was used to identify and characterise disulphide linkage in hirudin, insulin and lysozyme. Raman data provide new insights about therapeutic protein surface organization, which may serve as a basis for the design of therapeutic proteins.