My Proteincomplex (2 different proteins) is stored in Buffer containing 10 % sucrose. At a level below ~5 % Sucrose, the complex is precipitating.
I would like to perfom native MS to analyse the exact mass and stochiometry of the complex.
As far as I know, glycerol and other buffer additves (NP-40, Triton X ...) causes mass-additives and are not suitable for MS analysis.
If sucrose is not suitable either, are there other possibilities of keeping my protein in solution?
It's probably best to remove the sucrose. If you don't have any protein without sucrose, you can extensively dialyze a sample to remove the sucrose. Don't worry about the precipitate because it will likely be re-solubilized by adding 6-8 M urea, which I believe is OK for MS.
Hi,
You could consider coupling size exclusion chromatography to your mass spec and running that in 100 mM ammonium acetate, for example. In that way, you can 'desalt' your protein sample just before it enters the MS.
Good luck!
The best approach would be to talk to people who will run the MS analysis for you. Ask them what kind of instrument is available for this type of experiment and is it allowed or not to change the buffer system personallly for you. They will inform you also how you should prepare your sample. Urea is absolutely out. Maybe you could try MS-compartible detergent, like Rapigest or something else. It depends very much on what is allowed and accepted in a particular MS laboratory/service
Sorry for the late reply.
Don Kaiser, your solution wouldn't work. My protein complex is formed by non covalent interaction. By disolving the pellet in urea, I would loss the protein complex and will not be able to get the native mass. And this is, what I would like to know.
Eef Dirksen, we tried sec coupled with light scattering to get informations about the native mass, but the protein will precipitate, if the buffer conditions changes on the column or by dilution at sample preparation.
Natalia Battchikova, I am in close contact with the MS-guys, as I perform a lot of experiements at their lab. I will ask, if the detergenz you mentioned are possible,
Thanks for your answers. I will inform you, if I got some results.
Hi Christian - I assumed you had a relatively pure protein complex and the total mass of the two individual subunits would be the mass of the complex. But maybe it's not that pure or simple. Equilibrium analytical ultracentrifugation can help determine native complex stoichiometries.
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