I am not familiar with the details of your experiment, but concerning disulfide shuffling:

• Keeping the sample pH low (at or below pH 3-4) with acid should limit the formation of new disulfide bonds by keeping your free thiols protonated.  You can determine what you are willing to live with by looking up the pKa of Cys thiols.  Potentially, this could be simple and sufficient for all you need, but that also depends on what you are aiming at with your experiments, of course.

• Obviously, you have to run some tests first. You might want to test Bruce's suggestion (which is the more proper method) and see if you get the same results when you try acidic pH to control shuffling.  Alternatively, denature+reduce a cheap protein (lysozyme? BSA?) and see if you get any disulfides to recover with and without acid treatment.

Also, is the protein you are struggling with an RNase inhibitor by any chance?

I suggest blocking (alkylating) all the free disulfides bonds in the protein with something like 4-vinylpyridine then doing the analysis while avoiding adding any reducing agents like 2-mercaptoethanol see the link for details.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3607449/

Hi Chuanlong,

Reduction and alkylation is the only way to stabilize random disulphide bond formation in your sample.  Depending on the concentration of the protein, you will have to follow protocols to first reduce the cysteines followed by alkylation of the cysteines.

You will need to remove all the reducing and alkylation reagents before LC-MS/MS.  This is done by "desalting" of the protein and depending on the size of the protein, you can use  the appropriate reverse phase chromatography packings.  The reducing and alkylating reagents will elute from the reverse phase beads and leave the protein cleaned up; at this point, you can elute the protein using organic solvents, typically, acetonitrile.

Best,

Hediye.

I am not familiar with the details of your experiment, but concerning disulfide shuffling:

• Keeping the sample pH low (at or below pH 3-4) with acid should limit the formation of new disulfide bonds by keeping your free thiols protonated.  You can determine what you are willing to live with by looking up the pKa of Cys thiols.  Potentially, this could be simple and sufficient for all you need, but that also depends on what you are aiming at with your experiments, of course.

• Obviously, you have to run some tests first. You might want to test Bruce's suggestion (which is the more proper method) and see if you get the same results when you try acidic pH to control shuffling.  Alternatively, denature+reduce a cheap protein (lysozyme? BSA?) and see if you get any disulfides to recover with and without acid treatment.

Also, is the protein you are struggling with an RNase inhibitor by any chance?

 Thank you all guys for adding very helpful suggestions to me. I will try to prepare my samples under neutral or slight acidic condition with proper IAA alkylation on BSA first then to see if this condition also works to my protein.

Hi Ismet, the protein I am working with is not RNase inhibitor. It is a enzyme in NO signaling.