C-terminal Sequencing?

gvdries gonzalez.vandriessche at rug.ac.be
Thu Mar 30 06:23:04 EST 1995


Hi Mandy,

Yes it is possible to perform c-terminal sequencing. There are different
methods available, of which some may be useful to you. The first
technique, most comparable to classical n-terminal sequencing is
chemically. With the current state of the art, the amounts needed for this
are however so large (at least 3 nmol) that I would not recommend it.
Hewlett Packard has very recently launched the first commercial
instrument, of which I don't know if there are already sold. 
A more applicable technique is using carboxypeptidases. These enzymes
(purchased by Boehringer and other manufacturers) release one by one the
c-terminal residues. The released amino acids are than analysed by amino
acid composition analysis. In a time coarse experiment, you can follow the
order of release. Instead of amino acid composition analysis, you can also
take at several time steps a small amount of material and apply it to
matrix-assisted laser desorption mass spectrometry, a very sensitive and
acurate technique to determine the molecular weight. From the differences
of the MW  before and after digestion you can determine the released amino
acids. 

I don't know exactly who in the uk could do this for you. There must be
enough people having the techniques available. Otherwise you can try to
convince my boss, Prof. Josef Van Beeumen to let to perform the work here.
You can ask him by mail at the adress Jozef.Van Beeumen at rug.ac.be. 

hope this helps

Bart


Bart Devreese
Dept. of Biochemistry, Physiology and Microbiology
University of Gent
KL Ledeganckstraat 35 
9000 Gent, 
Belgium
(Bart.Devreese at rug.ac.be)








In article <udbl119.38.06B77978 at bay.cc.kcl.ac.uk>,
udbl119 at bay.cc.kcl.ac.uk (Mandy Johnstone) wrote:

> Hi folks,
> 
> I have a proteolytic fragment that is generated from endogenous calpain 
> mediated proteolysis of a brain protein and I have mapped it to the very 
> N-terminus using a polyclonal antibody which was raised to the N-terminal 
> residues. What we would really like to know however is what the
C-terminus is. 
> Is it possible and if so where can we sent the fragment for C-terminal 
> sequencing? Many thanks in advance.
> Mandy.
> 
> _______________
> Mandy Johnstone
> (M.Johnstone at bay.cc.kcl.ac.uk)
> 
> King's College, London.



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