Mary,
Perhaps you could take the peptide mixture (that has not been down the RP
HPLC column) and re-isolate the His tagged part using a nickel column,
just like the original purification that was used to pull-out the His
tagged protein. Clean the peptide up on RP chromatography--to get rid of
all of the imidazole--and to verify that you only pull out one tryptic
fragment (one peak on HPLC) and then sequence the peptide.
Another route would be through ion exchange chromatography:
Below pH 6.5 all of those His's (sp?) will be positively
charged.
Generate a table of all of the tryptic fragments from this protein,
including the N-term His tag sequence, and unless this is a very basic
protein, you will probably find that at pH 5.5 or so, the N-terminal
fragment is the most positively charged fragment that you have. This
would make it fairly easy to pull this peptide out of the mix using cation
exchange chromatography.
Another possible route:
On RP HPLC I often identify peaks that contain tryptophans and tyrosines
by their absorbance at 280--you can often tell by their relative peak
heights who is who when you take into consideration the extinction
coefficients of tyrosine and tryptophan (This is provided your digest
is indeed complete.) If you are lucky your first peptide has 280
absorbance a distinquishable amount of 280, so that might be a tool you
could use.
Deb McMillen
Institute of Molecular Biology
University of Oregon
Eugene OR 97403
On Fri, 17 Nov 2000, Mary Bower wrote:
> Dear colleagues,
>
> I have a protein digest which has been separated by HPLC. My customer
> suspects that there is a His tag on the N-terminal end of the protein, and
> would like to sequence the N-terminal fragment. Does anyone know of a
> paper that refers to a technique that enables you to identify such a
> peptide from a mix?
>
> Thanks in advance.
>
> Mary Bower
>
> Mary Bower
> Laboratory for Macromolecular Structure
> Dept. Biochemistry
> Purdue Unversity
> West Lafayette, IN 47907
>
> voice: (765) 494-6540
> fax (765) 494-7897
>
>
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