Radhika,
Its not clear if you are running both peptides at the same time, or
if a doublet shows up with each peptide run separately. If it is the latter
as I suspect, the following is a possibility to consider.
You might be getting cis/trans isomerization at one of the Pro
residues. This would explain why a purified peak produces the initial
profile when rerun, i.e. The purified peak is equilibrating with time to
the two conformers. If the Maldi says the mass is right, it seems that you
have sucessfully synthesized the intended peptides but those particular
peptides have the interesting property of adopting at least 2 relatively
stable conformations with relatively slow kinetics (minutes).
Greg
>Hi Colleagues,
> I have benefitted a lot form the discussions that go on in this group
>, and
>would like to avail of your collective wisdom.
> I am faced with a peculiar problem with the synthesis of two peptides that
>are designed to identify a immunological epitope. The sequences are as
>follows,
>TTVYPPSSTAK and TVYPPSSTAK.
>I have synthesized these two sequences twice on the Pioneer with two different
>batches of the acid Lys resin. My HPLC shows two peaks almost on the top
>of each
>other. We tried purifying the major peak on a prep column, but analysis of the
>"pure" peaks seem to indicate that nothing has changed. They look identical to
>the crude HPLC. The mystery is that the MALDI of both the samples do not
>indicate anything untoward. The only peaks are that of the peptide.
> Is it possible that one of the amino acids might be a racemic mixture? All
>my reagents are form PE Biosystems.
> Will AAA tell me anything if there were isomers.
> I have calibrated both my analytical and prep c18 columns.
>Thanks.
>Radhika.
>City of Hope, Duarte CA.
Gregory A. Grant
ggrant@molecool.wustl.edu
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