On-colum precipitation, storage, recovery

[DJ]

4C Storage of low-abundant protein/peptide samples often leads to substantial losses-- presumably through binding irreversibly to the walls of the tube on standing, while evaporation is also associated with irreversible sample loss.

What if a low-abundant peptide/protein sample were bound to a trap column, .mobile phase pushed or pulled out to dry the bed, then a few column volumes of an organic like ether or hexane is pulled through the trap, causing "precipitation" of the bound protein/peptide sample as a film on the support. Next, lyophilize and store at -80.

The idea is to temporarily store a low-abundant sample, such that oxidation and tube adsorption are minimized. The sample would then need to be recovered quantitatively from the dried trap. Approaches would depend on type of sorbent in the trap, but trifluorethanol, DMF, concentrated formic acid may be good candidates if all else fails.

Does anyone have any thoughts? Is the idea doomed to fail? What types of supports would make sense to allow in initial binding, precipitation, and recovery best? I was considering PS-DVB or polyhydroxyethyl.

Any thoughts?

[Peter Apps]

This is not really my area, but surely any of the reagents that you propose to get the proteins off the traps would be just as effective at rinsing them out of a tube ?

Peter

[HW Mueller]

Besides, there are books on handling proteins. Denaturing them out on a huge area is a very good way to oxidize them.

[DJ]

Not clear on the connection between denatured protein oxidation. So long as solvents de-gassed, column bed dried under vacuum, I don't know see where the protein would encounter oxygen.

I am hoping for guidelines as far as optimal pore size, bed type, etc for 5-10 kDa proteins or peptides. Assume for now oxidation can be prevented by working under inert atmosphere.

[HW Mueller]

Well, I thought that Peter´s comment would be sufficient to dissuade you from pursuing this, so I was a bit short cut. I meant to say that you will very likely thoroughly denature your material, and that significant oxidation can not be prevented on top of that. The claim on oxidation is based on my study on freeze drying serum and ascorbate radicals therein (published in British Journal of Cancer), where large surfaces play a role.

[DJ]

This is not really my area, but surely any of the reagents that you propose to get the proteins off the traps would be just as effective at rinsing them out of a tube ?

Peter

In my experience, when low-abundant sample is lost to the walls of a pp tube, it's gone for good. No washes, including HFIP, neat formic acid, etc can bring it off.

I was hoping that, perhaps if the sample were precipitated on a hydrophilic support with less surface area than a pp tube, that recovery (of the precipitated hydrophobic peptide) would be more promising.

[Peter Apps]

How about ordinary glass tubes ? - that will give you a hydrophilic surface.

Peter