"mike.naldrett" wrote:
> Deb
>
> Thanks for your quick reply - nice idea. We're looking into it as we just got
> the PCR product sequenced. First look suggested SER was spot on, but I think
> closer checking wouldn't go amiss.
Mike,
On inspecting the DNA sequence to reliably predict your protein sequence--if this
protein is expressed in E coli, we have often found ( not published) other amino
acid substitutions--usually as mixed sites. For instance, a VERY careful worker
last week was expecting YSL and when we did protein sequence analysis of the
protein we found 258 pmole Y, 156 pmole S with 80 pmole of N at the second residue,
and then 262 pmole of L--all you need is a bug that is even a little better at
putting N in place of the S. Some other mixed sites that I have observed are T and
K, N and T, and D and A. I'd love to know other mixed sites that have been
observed.
A tryptic map of your protein analyzed by mass (a MALDI with a mirror would work
well) to see if you can spot fragments that reflect the difference in mass between
Serine and Asparagine in the sequence. Unfortunately, I have found that often when
a bug does this mix at one site it does it at several other sites.
> have you any more info of acid- and
> base-labile sequences ie just the one good ref summarising them (or a summary
> you've drawn up)?
Here's my (old) list of acid/base labile bonds. The only reference
I have on my hand-written page is Methods in Enzymology Vol 47 p 145--but
I somehow think that it doesn't refer to all of these.
Acid labile
D-P this is the most labile of the lot, I think
G-P
D-G
Base labile
T-P
R-P
N-G
Then I have a note about the deamidation of Asn being common in the
sequences, N-G, N-S, and N-T.
Yes, let us know what you find! I like John Hempel's idea about the
phosphorylation (if this is a native protein and not expressed in E coli)
Deb McMillen
Institute of Molecular Biology
University of Oregon
Eugene OR 97403
>
>
> Thanks again
>
> Mike
>
> ------------------------------------------------------------
>
> Dr Mike Naldrett - Manager
> Protein Sequencing & Proteomics Facility
> John Innes Centre(NFL)
> Norwich Research Park
> Colney
> Norwich
> NR4 7UH
>
> tel +44-1603-450773 direct line **PLEASE NOTE THE CHANGE OF NUMBERS**
> fax +44-1603-450018
> e-mail: mike.naldrett@bbsrc.ac.uk
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