Lisa -
Hydrophilic Interaction Chromatography (HILIC) works well for acidic
phosphopeptides; see Boutin et al., J. Chromatogr. 583 (1992) 137-143. You
start with @ 85% ACN and run a decreasing ACN gradient. The mobile phases
should contain about 15 mM of some electrolyte. The original paper used
triethylamine phosphate, but you can use ammonium formate or acetate (you can
monitor at 270 nm, since all your peptides contain Y). If solubility is an
issue, you can dissolve your peptides in a minimal volume of
hexafluoro-2-propanol (HFIP) and then dilute them with @ 20 vol. of the
starting mobile phase. You can also include 50 mM HFIP in the mobile phases
to insure solubility if necessary. It's volatile and transparent, and far
preferable to guanidinium.HCl as a mobile phase additive if you can get away
with it. Loading capacity of a HILIC column is slightly less than that of a
comparable reversed-phase column.
Best regards,
Andy Alpert
PolyLC Inc.
9151 Rumsey Road, ste. 180
Columbia, MD 21045
tel: (410) 992-5400
****************************************************
<< Subj: PepSyn:solubilizing and purifying phospho peps
Date: 08/30/2000 9:24:34 PM Eastern Daylight Time
From: bibbs@scripps.edu (lisa bibbs)
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CC: peptide@scripps.edu, woodside@scripps.edu
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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