what a coincidence. I digested a longish peptide just yesterday with
Boehringer sequencing grade Asp-N. My digestion mixture was 5 microlitres
sample from an LC fraction (0.1% TFA, 30ish % acetonitrile), 5 microlitres
50mM ammonium bicarb, 0.5 microlitre Asp-N (a 2 microgram vial
reconsitiuted in 20 microlitres water), incubation at room temperature for
an hour. The resulting sequences are below, you can see the specificity
under these conditions can include both Asp-N and Glu-N.
The parent sequence is: SHSDSLDHDIFQLIDPDENKENEAFEFK
note that this is a tryptic fragment and the lys-glu bond is completely
resistant to trypsin.
The Asp-N products are:
DIFQLIDPDENKENEAFEFK
DENKENEAFEFK
DPDENKENEAFEFK
DIFQLIDPDENKENEAF
Note that the first three fragments are perfectly reasonable Asp-N products
whilst the fourth demonstrates the potential for a Glu-N specificity in the
protease.
It is interesting that the third product (DPDENKENEAFEFK) is predominantly
(by MALDI analysis) in a form 12u larger at 1725.3u than the predicted mass
at 1712.6u. I'm not sure of the Asp-Pro acid cleavage mechanism but I
wonder if the 1725.3u species is an intermediate along that path (my matrix
contains 2% TFA to neutralize the alkaline reaction conditions).
Any suggestions welcome.
regards to all.....Ken
>Is Asp-N sequencing grade from Boehringer cleaving in ammoniumbicarbonate
>and how is the specificity? Or is there another usable buffer for direct
>ESI-MS analysis?
>Thanks for answers!
>Greetings Daniel
>
>Dr. Daniel Hess
>Friedrich Miescher Institut
>R. 1066.3.04
>Maulbeerstr. 66
>CH-4058 Basel
>Switzerland
>
>Tel.: ++41 61 697 85 44 or ++41 61 697 68 68 FAX: ++41 61 697 3976
>e-mail: dhess@fmi.ch
********************************
Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy 3065 Victoria
AUSTRALIA
Telephone: 61-3-9288 2480
Facsimile: 61-3-9416 2676
Email: k.mitchelhill@medicine.unimelb.edu.au
Laboratory: http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRdocs/JHPSL.html
ABRF: http://www.abrf.org
***********************************