FW: breaking disulphide bonds (fwd)

bturner at caregroup.harvard.edu bturner at caregroup.harvard.edu
Fri Apr 27 15:33:38 EST 2001

Hello Michael,

If your problem is disulphide bonds, then you may want to try either
dithiothreitol (DTT) or Dithioerythritol (DTE), I think that they
are slightly better than 2ME at keeping protein sulfhydryls reduced.

Additionally, you may also want to consider blocking your reduced
sulfhydryls with either iodoacetamide, iodoacetic acid, N-ethylmaleimide
or 4-vinylpyridine (this last one might be more appropiate for subsequent
sequencing or MS).  Some disulphide containing peptides are notorious
for reforming if they are not covalently blocked or derivatized.

Finally you might also try using TCEP (tris(2-carboxyethyl)phosphine)
[I have included some references below].  According to the references
it is much more robust at reducing disulfides, i.e. active over a
much broader pH range, NO ODOR, not prone to disulfide reshuffling, etc.
It has actually been used to map disulfide linkages.  Some of the papers
below also contain references to Cys specific chemical cleavage methods.

You can find some more information about TCEP at the Pierce Chemical
web site [no affiliation]


If your protein contains methionines you could also consider chemical
cleavage using cyanogen bromide.

You may also want to get a copy of 

A practical guide to protein and peptide purification
for microsequencing 2nd edition
Paul Matsudaira editor
Academic press 1997
ISBN 0124802826

It has a lot of good suggestions and protocols.

Hope this helps,
Brad Turner

                    Bradley Turner
                Beth Israel Deaconess
                    Medical Center

Harvard Medical School          617-667-1215 phone
Division of Gastroenterology    617-667-2767 fax
Room Dana 605                   bsturner at biosun.harvard.edu
330 Brookline Avenue            bturner at caregroup.harvard.edu
Boston, MA 02215                bsturner at mbcrr.harvard.edu
                   Graduate Student 
      Molecular Biology, Cell Biology, Biochemistry
                  Boston University
                   bsturner at bu.edu

> ---------- Forwarded message ----------
> Date: Fri, 27 Apr 2001 11:46:57 +0100
> From: Michael Witty <mw132 at mole.bio.cam.ac.uk>
> To: methods at hgmp.mrc.ac.uk
> Subject: breaking disulphide bonds
> Dear All,
>         I am trying to fragment a protein for peptide mapping (ie
> N-terminal sequencing and Mass Spec), BUT the protein will 
> not fragment.
> I am using trypsin, chymotrypsin, elastase and V8 protease.  There are
> disulphide binds, but I have added 14mM mercaptoethanol.  My 
> question is:
> what are my options for more vigourous attempts at fragmenting the
> protein?  Am I failing (for some reason) to break up the disulphides?
> Thanks in advance for your opinions.  Mike.

TCEP References

1: Han JC, Han GY.
A procedure for quantitative determination of tris(2-carboxyethyl)phosphine,
odorless reducing agent more stable and effective than dithiothreitol.
Anal Biochem. 1994 Jul;220(1):5-10.
PMID: 7978256

2: Ren X, Kasir J, Rahamimoff H.
The transport activity of the Na+-Ca2+ exchanger NCX1 expressed in HEK 293
is sensitive to covalent modification of intracellular cysteine residues by
sulfhydryl reagents.
J Biol Chem. 2000 Dec 27 [epub ahead of print]
[Record as supplied by publisher]
PMID: 11134012

3: Lambert G, Traebert M, Biber J, Murer H.
Cleavage of disulfide bonds leads to inactivation and degradation of the
IIa, but not type IIb sodium phosphate cotransporter expressed in Xenopus
J Membr Biol. 2000 Jul 15;176(2):143-9.
PMID: 10926679

4: Katsumi A, Tuley EA, Bodo I, Sadler JE.
Localization of disulfide bonds in the cystine knot domain of human von
Willebrand factor.
J Biol Chem. 2000 Aug 18;275(33):25585-94.
PMID: 10831592

5: Dieguez-Acuna FJ, Woods JS.
Inhibition of NF-kappaB-DNA binding by mercuric ion: utility of the
reductant, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), on detection
impaired NF-kappaB-DNA binding by thiol-directed agents.
Toxicol In Vitro. 2000 Feb;14(1):7-16.
PMID: 10699356

6: Young Y, Zeni L, Rosenfeld RD, Stark KL, Rohde MF, Haniu M.
Disulfide assignment of the C-terminal cysteine knot of agouti-related
(AGRP) by direct sequencing analysis.
J Pept Res. 1999 Dec;54(6):514-21.
PMID: 10604596

7: Getz EB, Xiao M, Chakrabarty T, Cooke R, Selvin PR.
A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine
dithiothreitol for use in protein biochemistry.
Anal Biochem. 1999 Aug 15;273(1):73-80.
PMID: 10452801

8: Anderson MT, Trudell JR, Voehringer DW, Tjioe IM, Herzenberg LA,
An improved monobromobimane assay for glutathione utilizing tris-
(2-carboxyethyl)phosphine as the reductant.
Anal Biochem. 1999 Jul 15;272(1):107-9. No abstract available.
PMID: 10405300

9: Lundell N, Schreitmuller T.
Sample preparation for peptide mapping--A pharmaceutical quality-control
Anal Biochem. 1999 Jan 1;266(1):31-47.
PMID: 9887211

10: Wu J, Watson JT.
A novel methodology for assignment of disulfide bond pairings in proteins.
Protein Sci. 1997 Feb;6(2):391-8.
PMID: 9041641

11: Wu J, Gage DA, Watson JT.
A strategy to locate cysteine residues in proteins by specific chemical
followed by matrix-assisted laser desorption/ionization time-of-flight mass
Anal Biochem. 1996 Mar 15;235(2):161-74.
PMID: 8833324

12: Gray WR.
Disulfide structures of highly bridged peptides: a new strategy for
Protein Sci. 1993 Oct;2(10):1732-48.
PMID: 8251945

13: Herbert BR, Molloy MP, Gooley AA, Walsh BJ, Bryson WG, Williams KL.
Improved protein solubility in two-dimensional electrophoresis using
phosphine as reducing agent.
Electrophoresis. 1998 May;19(5):845-51.
PMID: 9629925


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