Lisa,
Try using 0.1M ammonium acetate or triethylamine acetate as your mobile
phase. Adjust the pH to 6-6.5 and run your normal acetonitrile
gradients. Acidic peptides like yours will probably be more soluble at
that pH than in a TFA mobile phase and the chromatograms may look more
normal. Also, don't be afraid to use guanidine buffers to dissolve your
peptides. At a mobile phase pH of 6 your sequences probably won't
precipitate on your column. The only downside to this method is that
you will need to lyophilize the purified fractions several times to get
rid of the salts.
Good luck
Russ
lisa bibbs wrote:
>
> Hello ABRF peptide guru's!!
>
> I have three peptides that have two phospho Y's each.
>
> One has overall -5 charge(19mer), -3 (19mer), and -2 charge (23mer).
> All precipitated during cleavage in their TFA. I have been able to
> get the one with -3 charge in solution at about 0.5 mg/mL slightly
> acidic water. The others are more like 0.1 and I have 0.25mMol Synth
> to purify.
>
> The HPLC traces look like a forest but the Mass specs are pretty
> clean. I got the -3charge purified on a c18 but when I run
> analyticals of the purified material, it reverts to multiple peaks
> again. I checked by MS of the various fractions I got from the prep
> and low and behold they have the same mass so I have multiple charge
> species that run at different retention times.
>
> I've thought about dissolving the other two in Guanidinium HCl but I
> am worried about them ppt'ing during purification on my column let
> alone the fact that I will probably get multiple species again
> although their analyticals look much better.
>
> Any purification suggestions would be appreciated.
>
> Thanks,
> Lisa
> Lisa Bibbs, Ph.D.
> 10550 N. Torrey Pines Rd.
> SP3
> La Jolla, CA 92039
>
> http://corefac.scripps.edu
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