Recently, I found that my protein of interest (Protein A) interacts with other proteins (Protein B) to form the complex.
In principle, protein A always exists as the dimer, so based on the molecular weight of the complex I guess that the complex is formed by 2 molecules of protein A and 1 molecule of protein B. But I need to confirm this speculation.
Please anyone having experience about this could suggest me the accurate experiment I should use for this purpose.
Thank you so much for all your help.
Best regards,
Hoang Thi Xoan
I think that depends on the type of methods you were hoping to use/ which of those are at your disposal. There are several different yet highly related techniques that are available for MS. I would consider taking a look at this article which is pretty general as a starting point for approaching your problem.
Comparison of label-free quantification methods for the determination of protein complexes subunits stoichiometry. Bertrand Fabrea, b, 1, Thomas Lamboura, b, 1, David Bouyssiéa, b, 1, Thomas Menneteaua, b, Bernard Monsarrata, b, Odile Burlet-Schiltza, b, , , Marie-Pierre Bousquet-Duboucha, b,
Best of luck,
Andrew
If the molecular weight that you have determined corresponds to the 2:1 stoichiometry, then that should be enough to prove the stoichiometry
In addition to mass spectrometry, explained by Andrew, the precise molecular weight of the complex could be determined by static light scattering or analytical ultracentrifugation. Gel filtration elution volume might not be reliable enough without additional proof.
If the subunits can be produced recombinantly, you could co-express protein A fused to tag X with protein A fused to tag Y. If protein A fused to tag X is co-purified via tag Y, then more than one molecule of protein A is present in the complex.
Hi there,
Protein crosslinking might also be helpful. In your case, if your complex is actually a trimer, you should be able to isolate it from SDS/PAGE (as well as AB heterodimer and A/A homodimer) and analyse the content of the bands by MS.
If you have access to someone who can run non-dissociating mass spectrometry, then this is an excellent technique to confirm the stoichiometry. A good recent reference is linked below - this describes detecting small molecules binding to proteins, so stoichiometry of two proteins should be OK.
Analytical ultracentrifugation is another good method, as Konrad suggested. Both methods can also detect heterogeneity, in case there are multiple stiochiometries - sometimes biology isn't as simple as we first think.
Good luck!
Article High-resolution mass spectrometry of small molecules bound t...
Hi, Try cross-linking and perform ITC to get the stoichiometry. Please have a look at our paper demonstrating the stoichiometry by many ways. Best wishes. Tham
http://www.jbc.org/content/286/30/26900.full.pdf
using a combination of static light scattering and an interactions software.... see attached. The Mass Spec methods will give very good data but suffer from the fact that it is no longer in solution, ITC stoichiometry will only give a ratio so will not differentiate between 1:1 and 2:2 binding.
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