I had good luck with pS peptides using 10 mM ammonium bicarbonate for
solubilization and purification. I used a "pH stable" C8 column from
Vydac and a linear gradient of acetonitrile in 10 mM ammonium bicarbonate.
Since the ammonium bicarbonate is volatile, it is fairly easy to get rid
of it. I don't know if you want ammonium ion as a counter ion for your
phosphate, but if it won't interfere with further studies, it may serve to
stabilize the pY.
Hope this helps.
Angela C. Murphy
On Wed, 30 Aug 2000, 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
>
*******************************************
Angela C. Murphy, Chemist
Lab. of Cell Biology, NHLBI, NIH
3 Center Drive, MSC 0301, Rm. B1-22
9000 Rockville Pike
Bethesda, MD 20892-0301 USA
tel.: (301) 496-2324
fax: (301) 402-1519
email: acmurphy@helix.nih.gov
*******************************************
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