Chromatography Forum

Hello,

I work in protein design lab and I have a new set of proteins that are mutants of a simple four helix bundle motif that is approximately 14kDa in size. The original protein takes to C18 RP HPLC 20 to 50% Acetontrile 0.1% TFA in fifty minutes swimmingly but this new set has some sticky issues.

The protein has 4 glutamates switch to alanines and one histidine replaced with cysteine.

When I run the new proteins I get a large amount of retention of my protein on the column so that when I run a blank run after a protein run I get the same peaks with the same A280 intensity. This can happen for multiple runs and after long 100% Acetonitrile washes.

I have moved to a C8 column and with the suggestion of the company I buy my columns from I added 50mM HFIP to my running solutions. I get much better intensity for my protein but I still have this retention problem.

My boss thinks it is the cysteine causing this problem.

Any help would be appreciated.

Free cysteine residues can cause severe tailing on silica-based materials in any mode of chromatography. This is evidence of some sort of strong interaction with the material independent of the other forces involved. We had terrible problems this way in analysis of glutathione (GSH). The problems disappeared when we switched to an HPLC column packed with a polymeric material. Try that.

Free cysteine residues can cause severe tailing on silica-based materials in any mode of chromatography. This is evidence of some sort of strong interaction with the material independent of the other forces involved. We had terrible problems this way in analysis of glutathione (GSH). The problems disappeared when we switched to an HPLC column packed with a polymeric material. Try that.

Totally disagree with this statement. When you analyze proteins or any compounds on mixed-mode column you don't have any tailing due to a shielding effect of basic group which is placed to the surface of the column (like in Primesep and Promix mixed-mode columns). Some tailing can come from the system and interaction with metal parts along the mobile phase flow.

Perhaps I should have been more specific. In our case we were using an anion-exchange column, to which reduced glutathione (GSH) was electrostatically attracted. This may have resulted in more intimate contact with the stationary phase than the mixed-mode material you're talking about, which I presume is a reversed-phase material with an embedded amine group. The only change we made was to switch to a metal column containing a polymeric anion-exchange material, in which case the severe tailing disappeared. I observed similar problems with silica-based reversed-phase column analysis of glutathione. Whether or not this tailing of -SH compounds is a problem presumably depends on the combination of column and analyte on a case-by-case basis.

Vlad: In the interests of Science, please run reduced glutathione over one of these columns you describe and let us know what the peak shape looks like.

I don't have glutathione, but if you send me a sample we can run few experiments in the interests of Science

Thanks for the help.

I have a mutant without the cysteine and I will express and purify it to see if the cysteine is my issue.

Also could I just add BME or DTT to the protein before injecting?

I presume you mean to ask if the DTT or BME's sulfhydryl- groups would saturate the relevant sites on the stationary phase, sparing the proteins' from this interaction. Nobody's studied this possible solution because nobody has systematically studied this interaction of protein -SH groups; the data in the literature is typically just a line or two in passing in a paper. The controlled run you propose with the des-Cys version would be great! Please let us all know what you find. If you do find that Cys- is implicated, then consider getting a different column material, per my and Vlad's exchange above.