Dear Pinaki,
I would be tempted to determine the amino terminus and the mass of the
protein by MALDI and subsequently by Quadrupole MS if the maldi answer does
not provide a clear answer! If the N-terminus is defined then the mass
variation of an amino acid (minimally 57 amu maximally 186 amu) should not
defeat your assignment. If this fails then by quadrupole MS (Micromass
Quattro II maximum entropy deconvolution and mass assignment to less than
2-5 amu) the mass should be well defined and the answer should be obvious.
You may want to do the mass analysis both ways so as to convince
yourself/colleagues that the same answer is obtained.
Cheers,
alex
PS: I am assuming that the protein is not post translationally modified in
other ways.
awb
----- Original Message -----
From: Pinaki Dutta <pdutta@umaryland.edu>
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Sent: Friday, April 14, 2000 2:45 PM
Subject: C-terminal sequencing
>
>
> Dear all,
>
> I have contacted a few of you personally based on the archives of this
> server, and I thank you for your efforts, but I still can't solve my
> problem of C-terminal sequencing. Perhaps the group at large will be able
> to offer their advice.
>
> I have a bacterial protein of around 100kDa that I believe is processed
> (several amino acids are cleaved off by an active serine protease
> catalytic site 800 residues upstream) at the C-terminus. I want to be
> able to determine the site of processing. So I am on the search for a
> facility who can perform this analysis.
>
> The protein can be electroblotted to either PVDF or nitrocellulose or cut
> out of a gel. Sorry, but it's not purified to 100% (it is around 80%
> pure). Someone mentioned using mass spect to do the analysis, but it may
> not be reliable for such large proteins. Tryptic digests also may not
> gaurantee results if the C-terminal fragment does not elecrtospray.
> C-terminal sequencing would be ideal.
>
> Many of the resources mentioned in previous e-mails no longer offer the
> service or are unwilling to take on new outside projects. The
> facility at the Univ. of Michigan was mentioned several times, but neither
> the e-mail addresses nor the phone lines working at this facility.
>
> I would greatly appreciate any input from anyone who can provide this
> service or knows of someone who still does. Please reply to me personally
> at pdutta@umaryland.edu . I don't know if I'm correctly registered on
> the list serv yet.
>
> Thank you.
>
> --Pinaki Dutta
>
>
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