<x-charset iso-8859-1>Try removing the glycerol first before doing your phosphatase treatment.
Something like a Filtron nanosep fast flow centrifuge concentrators (VWR
sells them) should work to remove the glycerol from the CIP. The small
volume ones hold about 600ul and flow pretty quickly. 30 minutes of spins
(3-4 washes) should remove the glycerol down to a workable level. For
peptides I was using lambda phosphatase which also was in glycerol. By
working at a higher concentration of tryptic digest (27-50 pmole/ul)I could
just add a small dilution of the enzyme, incubate and do a further dilution
before looking at the peptides via MALDI-TOF MS. Unfortunately with most
phosphorylated proteins you don't have the luxury of having that much sample
to get your peptides that concentrated. As many of us have learned (from
the test ABRF phosphorylated digest a few years back) those short
hydrophillic phosphorylated fragments can be difficult to capture on a C-18
resin. Some had suggested using PerSeptive's Oligo R3, which would require
you to pack your own tip. You might also try a different digestion enzyme
like Lys-C (WAKO), Asp-N or Glu-C to produce a larger, easier to capture
peptide. You could also try IMAC Ziptips to capture and elute a specific
peptide. For a small peptide I would for sure use Fe as the binding metal.
Using Fe you may not be able to recover larger fragments, especially those
containing multiple phosphorylations but you probably will recover the
smaller peptides, as well as a lot of Asp and Glu containing tryptic
fragments.
Gary Lange
gary.w.lange@pharmacia.com
Pharmacia
700 Chesterfield Parkway North
Mail Zone AA2I
St. Louis, MO 63198
636-737-6602
fx 636-737-7005
-----Original Message-----
From: liho2 [mailto:liho2@umdnj.edu]
Sent: Wednesday, May 23, 2001 10:24 AM
To: Recipients of ABRF List
Cc: Recipients of ABRF List
Subject: remove glycerol from phosphopepitdes/phosphoproteins
Dear All-
I am working on a phosphorylation site project. I've tried to use
phosphatase (CIP) to
determine the number of phosphates in the protein and in its tryptic
peptides. However, the
CIP we got from NEB is stored in 50% glycerol and 10 mM Tris buffer. My
questions are:
1. How to remove glycerol from the sample after incubating my protein with
CIP? I have tried
C4 ziptip without much luck at recovering my 30 kd protein.
2. How to remove glycerol from hydrophilic peptides derived from phosphatase
treatment of
the trypsin digest of the 30 kD protein mentioned above? I've successfully
used C18 ziptip
at recovering larger/hydrophobic peptides. But smaller/hydrophilic peptides
did not bind,
and are still mixed with glycerol in the 0.05% formic acid wash fraction
from the ziptip.
Thanks for your input!
Hong Li
MS Core Facility
NJ Medical School
Newark
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