From: "Juhani Saarinen" <jksaarin@operoni.helsinki.fi>
Date: Thu, 2 Jan 1997 17:21:28 EET DST
Subject: Re: LC MS/MS HELP!
To: Recipients of ABRF List <abrf@aecom.yu.edu>
> Date: Fri, 27 Dec 1996 13:44:22 -0700
> From: bibbs@scripps.edu (Lisa Bibbs)
> Subject: LC MS/MS HELP!
> Cc: forest@scripps.edu
> To: Recipients of ABRF List <abrf@aecom.yu.edu>
> I need some help or rather an investigator here does. This is my problem.
>
> The investigator has a 17 kDa protein that she believes has two
> modifications very close to each other (crystallography data). She
> believes she knows exactly what these modifications are. She needs this
> protein digested with Lys-C and then the fragment identified by MS and by
> sequencing, and then she needs a fraction of the peak for an assay.
>
> We have been unsuccessful in helping her. We digest the protein fine,
> purify, collect fractions and then send part of those fractions for MALDI.
> We have not been able to isolate the fragment of interest. I have tried
> just about everything. Our MS bill is skyrocketing.
>
> I thought with the new technology that is out there, someone could run LC
> MS and then when the mass of interest shows up, get the daughter ion info
> which should give us some sequence information. Since the investigator
> needs some of the peak for an assay, we need fractions collected.
>
> Can anyone help her? Our MS facility is not ready for this yet. She is
> really under the gun and needs this info ASAP.
>
> Thanks,
> Lisa Bibbs
>
>
Hi Lisa!
We had a similar case with a protein that came from our NMR lab. The
mass of the protein did not fit to the calculated MW within the mass
accuracy of our MALDI, and we wanted to check out the reason.
Protein was digested with lysC, and the total digestion mixture, as
well as the RP isolated peptides were analyzed by MALDI. All expected
peptides were found, except one, and of course all the peptide masses
obtained did fit to the sequence.
As a last desperate means we extracted the empty digestion tube with 20%
formic acid in isopropanol, and subjected the washing sample to the
maldi, and ha! there was the peptide. -wrong mass, and 2 changed
aminoacids.
Of course there might be several other reasons than this, but this is
worth checking out
best reg's
Juhani Saarinen
Protein Chemistry Laboratory
Institute of Biotechnology
University of Helsinki, Finland
e-mail jksaarin@operoni.helsinki.fi
Phone +358-9-708 59 414
Fax +358-9-708 59 416
Street adress:
Viikinkaari 9
FIN-00014, Helsingin yliopisto
PL 56