What we know about cysteine is that oxidized during the time? So, if it's the case, do you have any idea how stable cysteine mutant? I have already tried to incubate my protein sample into 1% DTT for 30 min at pH 7.5(if there is any oxidation). And I observed some differences, however, I have some doubt about the effect of DTT on my sample. Do you think the best thing to answer this question is mass spectroscopy or there is another way that ı could try and see?
Mass spectroscopy would be a good way to tell if the mass of the protein changed due to oxidation of the cysteine thiol. Another way to tell if the cysteine is in the thiol form is to test for its reactivity with a thiol-reactive probe. For example, after removing the DTT (or using TCEP instead, which wouldn't have to be removed), you could react the protein with fluorescein maleimide, which will react with thiol only. After removing the excess label, you could then tell if the protein became fluorescent. By measuring the stoichiometry of labeling, you could determine what proportion of the protein was in the thiol form during the reaction.
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