Dear Thomas, You did not mention if the sequence is very hydrophobic or highly charged. In case of a very hydrophobic peptide , with an high propensity to form beta strand, what you are seeing is not a dimer or a trimer, but a larger aggregate, somehow similar with what happen with beta amiloide peptide. By RP-HPLC, if this is the case, you would expect a broad peak. You should be able to detect this type of problem , if you have done Fmoc, when you try to dissolve your peptide after precipitation. We all know beast peptide , that require huge amount of 50/50 H2O AcCN to be solubilize. The fact that you are talking about a 8 mer of a 34 suggest me that you are dealing with a fragment of amiloide peptide. My suggestion is (especially since your customer maybe is thinking to do some aggregation experiments, ask him) to check the literature about amiloide peptide. I remember seeing an article from either vydac or HP (?) on a method to separate amiloid fragments using High Tempera!
ture
on HPLC (you would need a column thermostat, or at least an hot bath). They also reported a series of experiment with different solvent to solubilize this peptides. In my personal experience , after purificative RP-HPLC purification, the % of acetonitrile present in the fraction was large enough to prevent aggregation, especially if using a C18. I went directly to liophylization without evaporating AcCN. The recovered peptides can be stored under N2 and avoiding humidity. Little condensation on your peptide and it will solubilize , and start aggregate. I agree with Ping, mass should tell you something, but if you get on ESI, your mobile phase would be a 50/50 H2O/AcCN , and possibly disrupting the aggregation. Maldi can possibly tell you something, especially if you dissolve the peptide in water for deposition, but I would be careful, since the relatively high energy from the laser would disrupt the aggregate. Ultra filtration would be my gold standard to prove it.
Good luck
Matteo Villain
Thomas Andersen wrote:
> Hey ABRFers-
>
> I need some help.
>
> Do you believe that I should be able to separate an octapeptide from its dimer? And if so, by what technique?
>
> I have a well-characterized 8-mer that is just the niftiest little peptide you'd ever want to see, but upon storage in the lyophilized state, it loses activity. We have shown that there is no chemical modification of the peptide, and if we treat the stored peptide with urea, we can recover activity. Therefore, we think the peptide aggregates in storage. Also, we have shown that an earlier, larger version of this peptide, a 34-mer, does indeed aggregate in a very complex manner during storage in the lyophilized state. The 8-mer does not aggregate to a huge complex, so I think it is a dimer, maybe a trimer at largest. But I can see no resolution on high pressure gel filtration chromatography. I believe that I will never see much resolution, even if I hink around with pH, buffers, temperature, whatever. But I'd sure hate to be wrong about that. Do you think it is worthwhile trying harder to separate 8-mer from 16-mer? HPLC? Low pressure gel filtration? analytical ul!
tr!
> acentrifugation? Any other thoughts?
>
> Thanks!
> Tom
>
> Ø------------------------
> Thomas T. Andersen, Ph.D.
> Director, Protein Chemistry Core Facility
> Albany Medical College
> Albany, NY 12208
>
> 518 262-5368
> anderst@mail.amc.edu
-- Matteo Villain Telephone : ++ 41 22 702 5521 Research Assistant E-mail : Villain@cmu.unige.ch DÈpartement de biochimie mÈdicale University of Geneva Rue R.M. Servet, Geneva, CH.
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