Modified electrophoretic and digestion conditions allow a simplified mass spectrometric evaluation of disulfide bonds
Abstract
Proper formation of disulfide bonds in proteins is a prerequisite to their stability and function. Information on disulfide pattern may therefore serve as an indication of the proper folding of recombinant proteins, and can also be used in protein homology modeling for the purpose of structure refinement.
Protein handling and digestion at basic PH leads to disulfide bond scrambling. Here, we present a complete sample handling protocol, which allows processing of disulfide containing proteins at basic PH.
We modified the standard SIDS gel electrophoresis and protein digestion conditions by the addition of an oxidative agent, cystamine. This modification prevented disulfide scrambling, which we otherwise observed in the samples handled according to the general protocol.
Lysozyme from hen egg was used as a model protein for the development of the method.