Date: Tue, 21 Jan 1997 09:26:13 +1100
From: Ken Mitchelhill <Ken_Mitchelhill@muwayf.unimelb.edu.au>
Subject: Re: MS- CPY & MALDI
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Reply to: RE>MS: CPY & MALDI
Lisa,
I have had some limited experience with carboxypeptidase sequencing using both
the kit marketed by PerSeptive and with carboxypeptidases from other sources
(Boehringer Mannheim in particular).
The technique is quite straightforward but, like most enzymatic approaches, is
very sequence dependent.
For example, CPY doesn't particularily like C-terminal Lys or Arg, not
especially useful for tryptic or lys-c peptides, but you can usually get
around this problem using some CPB in the reaction as well.
Often the sequencing ladders will include a two residue gap, so much so that I
have had to extend my amino acid molecular weight table to include all amino
acid pairs.
The sequencing reaction will often stop at prolines and will always (in our
hands) stop at the residue c-terminal to a phosphorylation site.
With regard to peptide size, you want to try and keep the useful part of the
ladder out of the matrix so it is nice to start with MW 1000 to get say, five
residues.
With all these caveats, we still find this technique very useful in confirming
the identity of a peptide combined with other techniques.
Hope this is helpful....Ken
***
Ken Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute for Medical Research
41 Victoria Parade, Fitzroy 3065 Australia
Voice (03) 9288 2497 Fax (03) 9416 2676
ken_mitchelhill.medicine@muwayf.unimelb.edu.au
Maintainer of WWW home page for:
The Association of Biomolecular Resource Facilities
http://www.medstv.unimelb.edu.au/ABRF.html
***
--------------------------------------
Date: 21/01/1997 6:12 AM
To: Ken Mitchelhill
From: Lisa Bibbs
Hello Mass Spec people
My colleague has asked me to pose the following questions to you.
Thanks in advance for info.
Lisa
>Mime-Version: 1.0
>Date: Fri, 17 Jan 1997 14:54:47 -0800
>To: bibbs@riscsm.scripps.edu
>From: bothner@riscsm.scripps.edu (brian)
>Subject: question
>
>Hello Lisa
> Here is the question I have: Does anyone have experience with peptide
>sequencing using CPY and MALDI/TOF? I would like to know what size
>peptides work well (how large)? How many residues are you typically able
>to sequence using multiple dilutions of the CPY?
> Thank you, Brian
>
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Date: Sat, 20 Jan 1996 09:03:19 -0700
From: bibbs@scripps.edu (Lisa Bibbs)
Subject: MS: CPY & MALDI
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