Len,
I also have mixed results with the Ga IMAC Ziptips, but I still use them as
the first attempt at phosphopeptide analysis. Recovery typically tends to be
low from in-gel digests (less than 40%) even with 5-10 pmol of protein in a
band...at least in my hands. But note that the physical-chemical nature of
the peptide seems to have the most dramatic effect on recovery (some
peptides work others don't).
With some samples the IMAC ZipTips seem to recover nearly all of the
phosphopeptides and with other samples I may get virtually no recovery. In
my kinase substrate studies I sometimes use "scrambled" peptides....same
composition as the normal peptide substrate but different amino acid
sequence order. Occasionally the recovery of one of these phosphopeptides
is dramatically lower than for the other phosphopeptide. So it seems that
not only is the composition of the peptide (basic vs. acidic groups)
important but also the actual sequence ordering.
Despite these issues the IMAC ZIpTips are very convenient, hopefully
Millipore will develop even more chemistries for these tips....Protein A
ZipTips would go a long way in aiding Immunoprecipitation studies by mass
spec.
----------------------------------------------------------------------------
-------
Gordon Alton, Ph.D.
Lead Scientist
Assay Development and Analytical Protein Chemistry
Department of Informatics and Functional Genomics
Signal Research Division of Celgene
5555 Oberlin Drive
San Diego, CA 92121
Email: galton@signalpharm.com
Phone: 858-558-7500 x8252
Fax: 858-623-0870
WWW: http://www.signalpharm.com
----------------------------------------------------------------
-----Original Message-----
From: Len Packman [mailto:lcp2@mole.bio.cam.ac.uk]
Sent: Tuesday, February 13, 2001 2:53 AM
To: Recipients of ABRF List
Subject: IMAC ZipTips
I'd appreciate feedback from IMAC ZipTip users about how often they
are successful in isolating phosphopeptides from in-gel digest
material. My experience is that these tips work fine with beta-casein
but getting results from 'real' samples is not so easy, especially at
the subpmol level. I think there has been comment before about the
need to clean up the sample by reverse phase ZipTip before attempting
IMAC but that doesn't seem to solve the problem. I can confirm by
examining the IMAC flow through that known phosphopeptides have been
removed, but recovering them seems to be in the lap of the gods. I am
interested in subsequent LCQ analysis by nanospray, not maldi.
Any help/advice/experiences welcome and what are opinions on Fe vs Cu
, Ni or Ga ions?.
Len
-- ********************************************************************* Dr Len C. Packman Assistant Director of Research Department of Biochemistry University of Cambridge 80 Tennis Court Road Cambridge, CB2 1GA, UKTel: +44 (1223) 333639 FAX: +44 (1223) 766002 e-mail: l.c.packman@bioc.cam.ac.uk
Protein and Nucleic Acid Chemistry Facility http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html
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