Disulphide Bridge Analysis
Disulphide bridge analysis is one of a number of co-translational and post-translational modifications (PTMs) a protein/glycoprotein may undergo. The number and position of disulphide bridges present plays a crucial role in obtaining a correctly folded product which is crucial to its function.
Disulphide bridge and free sulphydryl group assignment is achieved using a strategy based on peptide mapping. Analysis generally involves digestion using a suitable enzyme or chemical and examination of the resulting mixture using LC-ES-MS to isolate and identify potential disulphide bridged peptides. These can then be confirmed by reduction and re-analysis using MALDI-MS and ES-MS or ES-MS/MS peptide sequencing. Using such powerful mass spectrometric techniques, enables even low levels of potential mismatched or scrambled disulphide bridge forms to be detected.
Disulphide bridge analysis is a key part of the ICH Q6B guidelines for the characterisation and confirmation of biopharmaceuticals in support of new marketing applications.