Lisa
Rule one in chromatography is to make them soluble in order to get the best
purification. The use of TFA is working against you here. You would be
better off at neutral pH using TEAP or TEAA on a silica column (pH 6.5-6.8)
or 0.1% TEA on a polymeric RPC column. The use of Guanidinium HCL is not so
good an idea since the silica columns will not do so well with it in there.
However, I suspect your peptides are eluting rather early, so you have a
loading problem in addition to a resolution problem.
Alternatively, the HILIC columns should be considered instead of the C18,
since the products of interest should come out last rather than within the
envelope of other impurities (if there are any in there). This means you
can load more sample and get great separations. With HILIC you can either
use a slight salt gradient (volatile ammonium formate if you have aromatic
residues) or a decreasing organic gradient to promote elution.
This should work quite well for you rather than struggling with other
techniques.
I made a posting a number of months ago about NOT using IMAC for
phosphopeptide isolation. I include it below for those of you who might
want to see it again.
Sincerely,
Amos Heckendorf
The Nest Group
-------
Subject: IMAC tips for phosphopeptides:
I would like to suggest that in the face of overwhelming interest in IMAC
for phosphopeptide isolation for protein digest work, IMAC (Please see
these papers on phosphopeptide isolation using the technique published by
Tempst et al, Anal. Chem. 1999 (71), 2883 or Neville et al, Prot. Sci. 1997
(6), 2436). ) as the sole microSPE clean-up tool is not the best sole
choice. As many of you have found out, the SDS in the gels poisons the
IMAC surface. Yes, you can wash out the dyes and SDS prior to IMAC
binding, but there remains a strong possibility that you may not have taken
out enough to leave sufficient binding capacity in the columns. The reason
that this is an issue is that in tip column microSPE, there is much less
packing material to work with and you reach the binding capacity limits
more easily than in a spin column or larger devices.
I suggest, in the face of your enthusiasm, that you consider instead the
use of HILIC, as Jen– et al. (Anal. Biochem. 215 (1993) 292) have shown
for the removal of SDS and salts from electroeluted proteins. The SDS and
dyes do not bind under the loading conditions and your phosphopeptides will
be the last to come out, since they bind more strongly than less polar
molecules. Then, if you want to sub fractionate these polar products, you
can pick out the phosphopeptides from this fraction with an IMAC tip with
better assurances of success.
While not completely foolproof, this seems more fool proof than the use of
C18, since with the C18 the SDS is retained longer than the peptides, but
not that much longer. If too many bed volumes of liquid are used, or too
much organic is used (ca. 45-65% MeCN), the SDS will elute along with the
peptides and contaminate the IMAC surface.
IMAC instructions are found at:
http://world.std.com/~nestgrp/pdf/Ap2/IMAC.pdf with general information on
these tips at: http://world.std.com/~nestgrp/pdf/Ap2/SuproTip.pdf.
Please feel free to call me if you need additional HILIC information or see
http://world.std.com/~nestgrp/pdf/pdf.html for the index of application
notes.
Sincerely,
Amos Heckendorf
-------
>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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