Hi all,
I am denaturing and refolding a protein complex containing disulfide bonds. After size exclusion and ion exchange, we're able to isolate what we think is correctly folded protein. I ran whole protein mass spec, and the parent mass is 5 daltons under the molecular weight. There are 5 disulfide bonds in the protein, so if they are all fully formed, I should see a mass 10daltons under the predicted MW, right? Thanks for the help!
Dear Chris,
You are absolutely right. You have 5 SH combine with 5 SH to form 5 S-S. Therefore, you will loose 5+ 5 =10 Dalton.
Rafik
Check out my C program. It requires a sequence (fasta format) and prompts you for the number of reduced Cys (assuming non-reduced are participating in disulfides).
Research Calculate the mw, aa composition, pI and extinction coef (28...
If I were you I'd bring my question to the local MS guru, to see if a combination of e.g. losing one N- or C-terminal residue plus some post-translational modification plus all disulfide bonds being formed (or not, or only some of them) could lead to the difference in mass you are detecting.
You may have to resort to digesting the protein and finding the disulfide-bonded peptides by MS. That will also tell you, in addition, whether the disulfides are scrambled or not.
Hi Chris,
are you sure that all cysteins did react? Try Ellmann's reagent or related to test whether there are free SH groups left. I fully agree with Alejandro's points.
Is there also a possibility that you may formed intermolecular disulfides which fragment during MS?
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