Jennifer,
As described your 100 to 200 cpm peptide is going to be very
difficult to work with. There just aren't enough counts unless you go for
very long scintilation times. Even then, its going to be hard to
distinguish from background. 1000 or more cpm would be better and it might
be wise to get more before going any further. Unfortunately, this is a
typical recovery from an in-vivo labeling and it may have taken quite a
while to get to this point in your purification. The other strike against
you is that a phosphorylated peptide is not going to run in the same spot as
the unphosphorylated peptide using most chromatography techniques. You're
probably out of luck if you want to sequence the mass portion of your sample
unless you are lucky enough to have phosphorylated mass too.
There is one technique, Manual Sequencing which you might find
useful. The reference for this technique is:
Sullivan, S. and T. Wong 1991. "A Manual Sequencing Method for
Identification of
Phosphorylated Amino Acids in Phosphopeptides." Analytical
Biochemistry 197, 65-
68.
This technique allows you to do protein sequencing at your bench. It
follows the radioactivity of the peptide and the released amino acid at each
cycle. When the phosphorylated amino acid is released, the peptide loses
its radioactivity and the solution the amino acid is in is now radioactive.
The elegance of this technique is that long scintilation counter times can
be used to follow low count levels. The disadvantage is that all we know is
radioactivity is released at a particular cycle, not the amino acid
identity. This is not a problem however, if one is certain about the
peptide's amino acid sequence. By knowing the sequence and the cycle of
release, the phosphorylation site can be identified accurately.
Richard Thoma
-----Original Message-----
From: fperini@unmc.edu [mailto:fperini@unmc.edu]
Sent: Thursday, July 20, 2000 12:56 PM
To: Recipients of ABRF List
Cc: Recipients of ABRF List
Subject: Re: ProtSeq
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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