Gene and Takeshi -
Coming to this discussion a bit late:
Can't help with prediction, but there is a method that's reasonably
convenient for ascertaining the disulfide bond locations. Digest your
protein with trypsin and separate the resulting peptides on a strong
cation-exchange column (our PolySULFOETHYL Aspartamide material works well)
with a salt gradient at pH @ 2.7-3. At this pH, the typical tryptic peptide
has a charge of +2, due to the N-terminus at one end and the Arg- or Lys- at
the other. Two such peptides linked by a disulfide bridge have a charge of
+4 and elute long after the rest of the peptides in the gradient. This way,
the selective isolation of the disulfide-linked peptides is easy. Phil
Andrews (U. Michigan) first introduced this method. Dan Crimmins (Washington
U. - St. Louis) has developed it with numerous applications. Here's a few
references:
1) Crimmins et al.: Anal. Biochem. 226 (1995) 355.
2) Crimmins, Meth. Mol. Biol. 36 (1994) 53.
3) Dong et al., J. Biol. Chem. 269 (1994) 6753 [determination of
disulfide bond locations in von Willebrand Factor multimers]
4) Dolmer and Sottrup-Jensen, FEBS Letters 315 (1993) [isolation of
disulfide-linked peptides from human complement component C3b].
5) Sandy et al., J. Biol. Chem. 265 (1990) 21108 [isolation of
disulfide-linked peptides from aggregating cartilege proteoglycan]
Finally, please note that this method works for isolation of tryptic peptides
crosslinked by means other than disulfide bridges. In Bendixen et al., J.
Biol. Chem. 270 (1995) 17929, plasminogen fragments were isolated that had
amide bridges formed by transaminase action on gamma-Glu and epsilon-Lys
groups.
Andy Alpert
PolyLC Inc.
9151 Rumsey Road, ste. 180
Columbia, MD 21045 USA
tel: (410) 992-5400 FAX: (410) 730-8340
*******************************************
<< Hi Gene,
Hello from Yokohama in JAPAN.
It looks late reply, and may be LITTLE informative for you.
I found
PROTEIN SEQUENCING PROTOCOLS Edited by Bryan John Smith
Methods in Molecular Biology Volume.64
ISBN 0-89603-353-8
In Chapter30,
Quintification and Location of Disulfide Bonds in Proteins
About LC-MS info. is NOT there.But Method for HPLC are there.
I also have strong interests to use your developing system.Regards,
Takeshi Fukuhara
tfukuhar@bio.titech.ac.jp
http://takeiteasy.4mg.com/
http://www1.bio.titech.ac.jp/~tkishimo/ref/ronbun-e.html
----- Original Message -----
From: "Gene Wijffels" <Gene.Wijffels@li.csiro.au>
To: "Recipients of ABRF List" <abrf@aecom.yu.edu>
Sent: Thursday, May 24, 2001 7:10 PM
Subject: disulphide bond prediction
> Hi ABRFers,
> I am trying to use protein digests and LC-MS to determine the disulphide
> bond arrangements within a couple of smallish knotty proteins.
>
> Does someone know of a web site that possesses a program that can
> predict/calculate/indicate the probable disulphide bonds of a (obviously
> non-reduced) protein given the mass of peptides from a digest, the
> protein's primary sequence, the endoproteinase specificity (and the buffer
> conditions of the digest if relevant)?
>
> I am aware of sites and programs that predict the mass of fragments and
> products of partial digests, but I need something smarter.
>
> Thanks heaps in advance,
> Gene
> Gene Wijffels
> CSIRO Livestock Industries
>
> Phone: (07) 3346 2510/14
> FAX: (07) 3346 2509
>
> Please note new postal address:
>
> CSIRO Livestock Industries,
> Level 3 Gehrmann Laboratories,
> Research Road
> University of Queensland
> St Lucia Qld 4072
>
>
> street address: Level 7
> Gehrmann Laboratories
> Research Road
> University of Queensland 4072
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