I do not believe that it is going to work.Phoshoserine and -threonine are
unstable,and
although you may find some,the PTH-amino acids will elute with the solvent
front.
In other words,it will be next to impossible to tell apart any PTH-a.a. and
free phosphate.
The normal strategy is to convert the phosphoa.a. to a S-alkylcysteine.I
used 1-propanethiol,while others have used ethanethiol,to yield
S-1-propylcysteine,and S-ethyl-
cysteine,respectively(see Meyer et al.FEBS
Letters(1986),vol.204,61-66.).The conversion is
done by beta-elimination in presence of an alkylthiol.The procedure works
well enough for
amino acid analysis,but the results look pretty ugly for sequencing.If Ser
is phosphorylated the reaction is only 1 hr,and the very specific
conversion from P-Ser to
a S-alkylcysteine has given me recoveries of >60% of a clean
fraction.Unfortunately P-Thr
is converted at a rate 20x slower,and I do not know of anyone who has done
sequencing.
I do not think that you should have any problem having three
phosphorylated sites.I have
never published anything on sequencing,because the peptides of interest had
both Ser and
Thr.You do not have to be concerned about separating any phosphorylated
from non-phosphory-
lated peptide,because you are interested only in the
S-alkylcysteine.Considering the low
amount of counts,I would not even think about it.
Jennifer Norton <jnorton@interchange.ubc.ca>@aecom.yu.edu> on 07/19/2000
11:20:48 AM
Sent by: Association of Biomolecular Resource Facilities
<abrf-request@aecom.yu.edu>
To: Recipients of ABRF List <abrf@aecom.yu.edu>
cc:
Subject: ProtSeq
Is there a protein sequencing facility that can process the following
request?
Thanks,
Jennifer Norton
>
>I am writing to inquire about the feasibility of sequencing a 32P-labeled
>(usually ~ 100-200 cpm) phosphopeptide purified from a trypsin-digested
>protein
>following peptide mapping on TLC plates, in order to identify the amino
>acid(s)
>that are phosphorylated in vivo. Importantly, the peptide itself contains
19
>amino acids (SDVWSYGVTMWEALSYGQK), and we suspect (STRONGLY!) that the
very
>first amino acid, a serine, is phosphorylated. What we need is a partial
>amino-terminal sequence of the isolated peptide to determine its identity,
>and a
>demonstration that the first amino acid contains a 32P phosphate group. We
can
>generate the purified radiolabeled peptide in less than a week, and send
it to
>you for analysis either in solution or lyophilized. Because of the
>complexity of
>the peptide and the number of potential phosphorylation sites (3 Ser, 2
>Tyr and
>1 Thr), we expect that it would be extremely difficult to identify and
isolate
>the correct phosphopeptide from the non radiolabeled protein. In fact, the
>mutation of the first amino acid (serine) to alanine causes the
>disappearance of
>three phosphopeptides on our peptide maps! It would therefore be most
>convenient
>to sequence the radioactive peptide directly. I have initially contacted
Sandy
>Kielland (she's the one who gave me your name) of the University of
Victoria,
>and she offered to do the experiment for us. Unfortunately, the sequencer
that
>she planned to use to map the 32P-containing amino acids suffered a lethal
>malfunction. Now, the best she can do is provide a sequence of the peptide
>that
>would contain "holes" where phosphorylated amino acids are located. It
>would be
>very important to us to get a "cpm count" for the 32P labeled amino acids.
>Please let me know if you can help us with this project.
>
>Sincerely,
>
>ClÈment Couture, Ph.D.
>Terry Fox Molecular Oncology Group
>Lady Davis Institute for Medical Research
>Sir Mortimer B. Davis Jewish General Hospital
>3999 Cote Ste-Catherine Road
>Montreal, Qc, CANADA
>H3T 1E2
>
>Phone: (514) 340-8222, ext. 3537
>FAX: (514) 340-7573
>e-mail: ccouture@ldi.jgh.mcgill.ca
>
Jennifer Norton, M.Sc.
Peptide/Protein Sequencing and Peptide Mapping
Nucleic Acid Protein Service (NAPS) Unit
Biotechnology Laboratory
University of British Columbia
Room 237 - Wesbrook Building
6174 University Boulevard
Vancouver, BC, Canada
V6T 1Z3
Tel: (604) 822-9688
Fax: (604) 822-0676
jnorton@interchange.ubc.ca
http:/www.biotech.ubc.ca/services/naps/
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