Disulfide bonds in cytoplasmic proteins

[Stephan Urban]

Dear collegues,
I have analyzed an insect cell derived recombinant protein with 10 cystein residues by high 
resolution mass spectrometry. The protein has been fused to a hexahistidine tag and was 
purifyed under denaturing, but non reducing conditions (6 M guanidine) on a nickel chelate 
affinity colum. The molecular mass that I´ve found was exactly 10 Da lower than the calculated 
mass; no multimeric forms could be detected. Addition of 10 mM 2-mercaptoethanol to the 
column buffers resulted in a mass that exactly matches the calculated one. Additionally 5 
further peaks with mass differences of 1-5x 78 Da, representing adducts of 2-mercaptoethanol 
were also found, showing, that reduction occured only partially. My conclusion from this 
observation was, that all of the ten Cys residues were intramolecular linked within the cell 
before purification; otherwise one should expect some multimeric forms. As the protein forms 
"inclusion bodies" in the cytoplasm this protein would be bridged by disulfide bonds in the 
reducing milieu of the cytosol.
Does anybody has observed similar things or knows anything about other proteins that are 
oxidized in the cytosol?

Thank you very much for your help

Stephan