"Recipients of ABRF List" <abrf@aecom.yu.edu>
References: <82136906C193D311976500A0C9EAD0487E388D@ems1000-03.monsanto.com>
Subject: Re: remove glycerol from phosphopepitdes/phosphoproteins
Date: Fri, 25 May 2001 09:09:02 +1000
MIME-Version: 1.0
Content-Type: text/plain; charset="iso-8859-1"
Content-Transfer-Encoding: 7bit
X-Priority: 3
X-MSMail-Priority: Normal
X-Mailer: Microsoft Outlook Express 5.50.4133.2400
X-MimeOLE: Produced By Microsoft MimeOLE V5.50.4133.2400
Message-ID: <OE15APi3BWZVo9DFw5L00006d1a@hotmail.com>
X-OriginalArrivalTime: 24 May 2001 23:09:10.0124 (UTC) FILETIME=[8A3B9AC0:01C0E4A6]
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Sender: Association of Biomolecular Resource Facilities <abrf-request@aecom.yu.edu>
Precedence: bulk
Errors-To: abrf-request@aecom.yu.edu
Hi Gary ;-)
all good suggestions, the point about small hydrophylic peptides is quite
salient especially given many protein kinases preference for hydrophylic
patches on substrates.
The other option to reducing the glycerol problem is, of course, to change
phosphatases. All my phosphatase work was done using alkaline phosphatase
that was supplied as lyophilised powder (Boehringer), no need to worry about
buffer components, just supply your own. I made up 1mg/ml in distilled water
and diluted into the required ammonium bicarb concentration.
For detailed protocols, see Mitchellhill and Kemp: Phosphorylation site
analysis by mass spectrometry in Protein Phosphorylation: A Practical
Approach, Second Edition, Edited by Grahame Hardie 1999.
Kindest regards....Ken
----- Original Message -----
From: "LANGE, GARY W [R&D/1005]" <gary.w.lange@pharmacia.com>
To: "Recipients of ABRF List" <abrf@aecom.yu.edu>
Cc: "Recipients of ABRF List" <abrf@aecom.yu.edu>
Sent: Friday, May 25, 2001 12:29 AM
Subject: RE: remove glycerol from phosphopepitdes/phosphoproteins
> 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
>
>
>
This archive was generated by hypermail 2b29 : Wed Nov 21 2001 - 14:16:55 EST