Re: tryptic digest

Marcus Macht (macht@sg17.chemie.uni-konstanz.de)
Thu, 19 Nov 1998 14:44:03 +0100

On Nov 19, 8:37am, Peter Hunziker wrote:
> Subject: tryptic digest
> long time ago my supervisors teached me that trypsin does never cleave the
> peptide-bond between LYS/ARG and PRO. Now, I also tell my students that
> this bond is not cleaved at all. However, in a recent LC/MS analysis we
> measured the masses of two peptides that indicated a tryptictic cleavage
> exactly at this bond (Enzyme: mod. trypsin from Promega). Since I could not
> believe it we additionally sequenced the peptides by Edman degradation and
> it turned out that trypsin has cleaved the LYS-PRO bond.
> On the C-terminal side of the cleavage site there were a number of GLY
> residues. Does anybody know if this may influences the specificity of the
> enzyme? Were my supervisors wrong and trypsin does cleave LYS/ARG-PRO bonds?
>
> best regards
> Petre

Dear Peter,

I had a very similar problem with unmodified trypsin from Sigma. The protein I
used to work on contained a sequence ...EEFKPKPRPFMPN... and after tryptic
digest and affinity chromatography I observed several peptides whose masses
could be assigned to trypsin-specific cleavages at the second and third basic
residue within the KPKPRP sequence. The software I was using (GPMAW) also does
by default not consider a tryptic cleavage N-terminal of Pro. I asked Peter
Hojrup from Odense university (who is the author of the programm) for some
literature for the cleavage resistance of X-Pro bonds. He gave me some
citations but not all of them mention this fact. E.g. in "Proteolytic enzymes"
by Beynon and Bond there is no mentioning of this resistance, while in G.
Allens "Sequencing of Proteins and Peptides" there is.
In my opinion, normally a cleavage before Pro will be more or less unlikely.
However in special cases such as special sterical conditions or anything else
it might occur in remarkable extent (in my case these had been the only signals
observed!).
So maybe your your supervisor had been correct for the usual cases while there
are some exceptions from this rule of thumb.

Yours sincerely,

Marcus

-- 
* Marcus Macht, AG Przybylski, Faculty of Chemistry, University of Konstanz
* Box M 732, 78457 Konstanz, Germany
* Tel:++49-7531-883391 / Fax:++49-7531-883097
* Email: macht@sgiclu.chemie.uni-konstanz.de
* URL: http://www.ag-przybylski.chemie.uni-konstanz.de/~macht