I have a system that generates carbonyls and dicarbonyls. I am trying to see what effects these carbonyls will have on cellular proteins. I am not looking for common AGEs, HNE or other chemicals like glyoxals and Acrolein, but a totally new mix of carbonyl species. Is there a method to detect general protein-carbonyl adducts?
Please find the method in this paper:
http://www.ncbi.nlm.nih.gov/pubmed/11514102
Dr. Banerjee, I don't want to detect oxidized proteins, or proteins with carbonyl groups on them after oxidation. I am looking for adducts formed between free floating external carbonyls and proteins. This will probably involve formation of a Schiff base between the floating carbonyl and amino group of some amino acid on the protein. I don't know any chemistry and have not yet read your paper completely, but I guess you derivatized carbonylated proteins with DNPH which will detect carbonyls in general, not Protein-Carbonyl adducts.
Hi Steingrimur,
I can not use carbon isotope in my system because the synthesis is very tough and the isotope containing compound might not behave the way I want it to. One of the cellular compounds will break down in to carbonyls in response to radiation. I know the identities of the monomers of original compound, but what kind of carbonyls are formed is not at all clear. Only evidence I have is detection of triplet carbonyl energy from the system in the form of luminescence and physiological effect of this energy, AND few predictions of the structures of expected carbonyls.
Hi Sanjay, fair enough.
Do you have a model system, e.g. using your system to modify a single protein, like BSA? Also, do you have access to an NMR that can detect 13C chemical shifts? 13C is naturally occuring (~ 1% of all carbon).
Hi Steingrimur,
Yes, I can design a system to first put in a known protein like BSA and then try to detect adducts using NMR and/or Mass Spec. Are you thinking of first running native unmodified BSA on Mass spec /NMR and then the one from my system and look for mass change, and/or structure change? I guess this is a good idea. I can then look for the adducts of BSA with the hypothesized carbonyl structure which should also tell me if the hypothesized structure (s) is true or not.
Do you think this should work? I am not at all familiar with NMR / Mass spec, but we have a central facility and those guys should be able to do it.
Dear Sanjay Premi,
Yes you are correct. Sorry for my misunderstanding.
Dr. Banerjee, it is alright. 99% of the people whom I ask the question suggest me the same because it is a bit confusing. By the way, I had already read your paper long back when I started searching for the methods. It is a brilliant study.
Thank you
Hi Sanjay,
Yes, I was thinking along the lines of doing trypsin digests of your modified vs. unmodified protein and obtaining a mass spec. profile. If there are different peaks in the modified protein. You should be able to deduce from their mass if they might have extra carbonyls. (You could even use a simpler protein, say, insulin or small peptides with known sequences)
Then you can use 13C NMR to verify that they have a signature consistent with carbonyls.
I'm more familiar with 1H NMR, but if your carbonyls are stable, I think you have a good shot.
Good luck!
Steingrimur,
Thank you for the helpful discussion. I will start on this one soon and let you know if I get anything.
Best wishes
Sanjay
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