IMAC for phosphopeptides or not?
nestgrp at world.std.com
Fri May 19 08:46:57 EST 2000
Subject: IMAC tips for phosphopeptides or not?
Dear 7tms_r newsgroup members:
As you are aware, there are several vendors for microSPE tips and spin
columns (ZipTip(tm), SuproTip(tm), TipColumns, MiniSpin(tm), etc.). I
present this here for the purposes of discussion only. We will all
hopefully benefit from a open sharing of information.
I would like to suggest that in the face of overwhelming interest in
IMAC for phosphopeptide isolation for protein digest work, IMAC 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 Jenö 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.
Once the IMAC is contaminated, it is dead.
While not completely foolproof, this seems better than the use of C18.
With C18 materials 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.
I hope this helps refine your thinking and I welcome input from the
group as to how you are solving this application.
The Nest Group
For examples on HILIC and microSPE protocols, see:
More information about the Proteins