Peptide problem

[chichibabin]

Dear All,
Please see the the trace attached.



The multiple peaks you see are in fact one peptide which is of a very high purity (when I run a much smaller amount of sample on an analytical column there is only one major peak with a little roughness around the edges). The peptide is insoluble and consequently it is loaded in a sample solvent based on pH 6 phosphate buffered 8M Urea. The HPLC mobile phase is H2O/MeCN 0.1% TFA and the method consists of a linear gradient. I would say the peptide is agragating in some way. Does anyone have any suggestions on how to improve the elution profile?
Regards,
Sat

[Uwe Neue]

This could be related to the difference in the sample loading pH and the chromatographic pH. Such problems can be solved by a trick that is called at-column dilution and was developed by us. Published in Chromatograpia 57 (2003) S-121 to S-127. If you contact me, I will send you a copy.

[HW Mueller]

Also, what do you get if you inject the peptide solvent (pH 6, 8M urea + ??) without the peptide?
Your statement of "high purity" is based on your analytical result?
Is this a synthetic or natural (isolated...) peptide?

[chichibabin]

Also, what do you get if you inject the peptide solvent (pH 6, 8M urea + ??) without the peptide?
Your statement of "high purity" is based on your analytical result?
Is this a synthetic or natural (isolated...) peptide?

With sample solvent alone no peaks are obserevd except at low retention time.
The high purity statement is based on SDS-PAGE primarily.
The peptide is formed by cleaving a precurser protein from a bacterial source.
Sat

[HW Mueller]

Nothing above 12 min(?) without the peptide in the solvent? I am assuming that this and your analytical technique are reverse phase methods, which are generally used to check on homogeneity (purity, if you will) of electrophoresis bands. If you assume that a peptide/protein of natural origin is not homogeneous you have a great chance of being on the correct track.
On improvements: If you have been looking in on this forum for some time you would already know my suggestion on this: Get rid of the TFA and use a buffer instead, phosphate if you don´t do MS.
If I am right on the molecular homogeneity than you should even get more peaks if you improve the method.

[chichibabin]

I have dialysed the peptide in the sample solvent (20mM phosphate pH6, 5M urea, 1mM EDTA) against cold water overnight. This precipitates the protein out of solution. I then centrifuged the peptide and removed the water and dried it. I then dissolved the residue in a phosphate pH 8 buffer with 6M GdnCl. This has solved the problem. I'll post the new trace soon.

[HW Mueller]

The "highly pure" peptide has enough protein in it that you could precipitate it out? How do you know that the peak you now get is your peptide??

[chichibabin]

I know the peptide is pure from SDS-PAGE. I also kknow its not soluble in water (only soluble in at least 5M urea). Therefore anything which precipitates when dialysed against water must be the peptide. I am performing mass spec to confirm this later today.
Cheers,
Sat

[HW Mueller]

This is the second case within a week which promts the recommendation to read posts again: Protein or peptide? Highly pure on basis of SDS-PAGE?

[chichibabin]

Sorry you've lost me.
I will clarify. I express a fusion protein in bacteria consisting of a chitin binding domain an intein and my polypeptide sequence (40 residues) at the N-terminus. I purify this fusion protein using a chitin resin with an FPLC system. If I add thiol to the column I can cleave off the polypeptide sequence and generate it as a C-terminal thioester. (For more details see NEB IMPACT expression system). Why do I want my polypeptide as a C-terminal thioester? So it can undergo a Native Chemical Ligation reaction (a stereospecific peptide coupling reaction) with an N-terminal Cys peptide. After the cleavage I elute off the polypeptide thioester in buffer I mentioned (phosphate pH 6, 5M urea...). It is this eluted polypeptide sample which I run on a gel and see as a pure band of 6kDa. It is this polypeptide that I dialysed against water.
Sat

[HW Mueller]

I referred to the following:
[quote="chichibabin"]..... This precipitates the protein out of solution. I then centrifuged the peptide and removed the water and dried it.

And,

my post from Sept 7, 6:43pm, where I state that it is unlikely that an electrophoresis band of a natural peptide would yield a pure substance.

There I also gave a suggestion, but, as we still do not know what type of HPLC you did to get your posted chromatogram it is not possible to give further advice. Anyway, you think that you have solved your problem.....
or are you waiting for the MS? How will the MS show that you had a pure substance giving rise to your chromatogram in your first post?