Modified electrophoretic and digestion conditions allow a simplified mass spectrometric evaluation of disulfide bonds
Abstract
Here, we present a complete sample handling protocol, which allows processing of disulfide containing proteins at basic pH. We modified the standard SDS 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.
We then applied our protocol to human leukocyte antigen CD69, for which the disulfide bonding is known, but only for its monomeric form. In addition, the disulfide arrangement was then 'de novo' identified in the recombinant murine leukocyte receptor NKR-P1A and in the larger glycosylated proteins beta-N-acetylhexosaminidases from Aspergillus oryzae and Penicillium oxalicum.