Tomas,
I have found in my LC/MS experience that a reverse phase column ( 0.1% TFA,
ACN gradient, C18 or C8) works very well to seperate dimers and trimers of
peptides of this size. I have seen dimers and trimers of peptides up to 40
mers with minutes of flat baseline between. The only catch is the
multimers have to be covalent. The non-covalents will not survive the ACN.
My experence is that noncolavents will not survive in the conditions needed
to run a size exclusion column under conditions that give decent resolution
to peptides of this size. Usually people have to put in ACN or high salt
to get the resolution and this tends to destroy the noncovalent
interactions.
I would explore addditives to the peptide during lyophilization to
prevent the multimer formation. Try maltose or another non-reducing sugar.
Mike Knierman
Thomas Andersen <AndersT@mail.amc.edu>@aecom.yu.edu> on 05/31/2000 09:39:14
AM
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Subject: peptide size
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 ultr!
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
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