My protein form dimer trimer and tetramer, these species forms as a result of disulfide bond formation from monomer. Confirmed by nonreducing and reducing SDS PAGE. My protein monomer contains 6 cys residues. I want to know which number of cysteine from the primary sequence are involved in the disufide bond formation by mass spectroscopy. I know the primary aa sequence.
The method is to digest the protein to completion with MS-grade trypsin, then to identify from the masses of the resulting peptides which are crosslinked. If there are ambiguities remaining because of peptides containing more than one cys residue, the use of a second protease, such as chymotrypsin, can help to resolve them.
You may use a simple and high-throughput method for the identification of disulfide-containing peptides/protein utilizing peptide/protein-matrix adducts. Some commonly used matrices in MALDI mass spectrometry were found to specifically react with sulfhydryl groups within peptide/protein, thus allowing the observation of the peptide/protein-matrix adduct ion [M + n + n′ matrix + H]+ or [M + n + n′ matrix + Na]+ (n = the number of cysteine residues, n′ = 1, 2, … , n) in MALDI mass spectra after chemical reduction of disulfide-linked peptides/proteins. Among several matrices tested, α-cyano-4-hydroxycinnamic acid (CHCA, molecular mass 189 Da) and α-cyano-3-hydroxycinnamic acid (3-HCCA) were found to be more effective for MALDI analysis of disulfide-containing peptides/proteins. Two reduced cysteines involved in a disulfide bridge result in a mass shift, so the number of disulfide bonds could then be determined, while for the other matrices (sinapinic acid, ferulic acid, and caffeic acid), a similar addition reaction could not occur unless the reaction was carried out under alkaline conditions. In general, this method is fast, effective, and robust to identify disulfide bonds in proteins/peptides. for more information you may go through this link http://www.sciencedirect.com/science/article/pii/S1044030509006722
Thanks all,
I want to do disulfide mapping of the protein dimer. I can see dimer band in non denaturing gel. Does it oky if i elute dimer from the SDS PAGE and use it for disulfide mapping. because some of the literature says, disulfide scrambling may occur due heating of the sample. And heating with loading dye is essential for SDS PAGE
In that case, as suggested by Adam B. Shapiro, you should go for a protease analysis where your protein is digested into small peptides. Then you should run the resultant mixture using LC-MS to identify the possible cleavage products. Based on this data and your prior understanding of the possible protein cleavage products depending on the sites of protease activity, you can find the odd one out very easily.