Hi all,
I am doing a small project in which I see that a mutant in protein A, that changes arginine to cysteine, can form a disulfide bond with another cysteine protein B. The problem is that protein B has 26 cysteine residues. I am appreciated if you could suggest some methods to identify which cysteine in protein B is responsible for the formation with cysteine in protein A? Thank you
Hi there,
I think the following article may help, it's about LC-EC-MS technique:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4686567/
This could end up being quite a complex mixture with multiple pairings involving relatively large proteins. Are there any aggregates and do you have access to LC/MS/MS?
Yes, that's how it is traditionally done. Classically the resulting peptides are analyzed both before and after reduction and alkylation and the reduction can be done electrochemically as referenced above. The potential for a complex mixture occurs because one of the proteins you described has 26 cysteines. Just for that protein alone imagine how many intra-protein disulfide combination could exist and those combinations each get digested into many fragments, the majority of which are two peptides linked by disulfide bridging. This multi-cysteine protein might also form disulfide-bridged aggregates. But then you're adding another protein which could possibly disulfide bridge in different combinations to a cysteine in the first protein. This second protein may also disulfide bridge to itself. Likely the reverse phase HPLC chromatogram will have a tremendous number of peaks to sort through by LC/MS and then certain MS peaks might need to go through another MS to identify it. The analysis might be simplified if there is some preliminary purification to remove aggregates or separate various isoforms from each other. You might want to do a literature search on cysteine pairing assignments to see how complex the digested sample might appear for multi-cysteine proteins. One that I worked on is Osteoprotegerin. Is the protein you're working with an antibody or something that has covalent subuntis?
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