>Any thoughts on the use of Ziptips to possibly clean up a tryptic digest
>of an electro-eluted sample from SDS PAGE? The sample was sent from Peru-
>highly coloured with Coomassie and I now need to separate the resultant
>peptides on my PE ABI173 cHPLC and am loath to inject such a mess.
>
>Any thoughts/protocols on cleanup of Coomassie from a peptide containing
>liquid would be greatly appreciated.
Suzanne
The "problem" with the use of SPE to clean up anything is that there is a
capacity issue we need to keep in mind. We all want our tools to be
"bullet proof" in their operation. In order for this to occur we need to
add capacity to the SPE tip to cope with the unexpected stuff in the
sample. This will add volume to the elution and thus we begin on the road
to discontent.
In SPE either the sample binds or it doesn't. We are not doing elution
chromatography as you do on an HPLC column. You select the mobile phase to
either stick it or to release it. We do not want to dribble it off (I'll
spare all of you the metaphors).
If it is possible to remove the Coomassie Blue before the digestion I would
encourage its consideration. Otherwise, you have two non-polar nuisance
species occupying the space that the peptides should occupy on the RPC
tips. Thus you would have to process the whole sample and recover from the
sample vial (assuming you cycled the waste into it) everything that did not
stick, in order to be successful. Not the best first choice for solving
this, and thus the reason desalting with C18 is less successful on certain
tips when taking samples from SDS gels.
Alternatively, removal of SDS and Coomassie Blue is easy with HILIC tips
and high organic conditions. The Coomassie Blue should even be left behind
in the sample vial during the loading step (see Paul Jeno's paper in Anal.
Biochem. 215 (1993) 292.). The peptides should bind under 95% MeCN loading
and elute at ca. 10% or less MeCN as noted in Alpert's posting. However,
in spite of my agreement with him, the concern I have with this suggestion
with regard to SPE is that there will not be enough capacity, since 25 ng
loading on some tips is normally the limit. It doesn't take much other
stuff in there to make your peptide the last in line to bind so be careful
to select a HILIC tip with enough resin there to do this. Fortunately
there are several choices in this regard, or as Alpert suggests, you can
always do it by HPLC directly into your MS.
Call me if you would like to discuss this further.
Sincerely,
Amos
Amos Heckendorf, Ph.D. (nestgrp@world.std.com)
The Nest Group, Inc., Value Added Resellers of HPLC Columns (VydacÅ,
PolyLCÅ, BioChrom HydrocellÅ, Jordi-GelÅ, Macherey-Nagel NucleogenÅ,
Higgins Analytical HAISilÅ100,TARGAÅ & CLIPEUSÅ, and Nest Group MaccelÅ)
Genomic SolutionsÅ PAGE mini-gels; and AmiKa COZAPÅ gel destaining pads,
BioDialyserÅ, Dispo-BioDialyserÅ , 96-Well Dialysis or 96-Well Equillibrium
Dialysis Plates, and SuproTipÅ MS MicroSample SPE Tips.
Tel: 800-347-6378 or 508-481-6223; FAX 508-485-5736;
45 Valley Rd, Southboro, MA 01772-1306
Applications and Protocols at:
http://world.std.com/~nestgrp/protocols/protocol.html
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