From: "Jay W. Fox" <jwf8x@virginia.edu>
Subject: RE: High Sensitivity MS/MS Sequencing
Date: Fri, 3 Jan 1997 08:57:10 -0500
To: Recipients of ABRF List <abrf@aecom.yu.edu>
----------
From: M. Hackett[SMTP:mhackett@u.washington.edu]
Sent: Thursday, January 02, 1997 12:40 PM
To: Recipients of ABRF List
Cc: Recipients of ABRF List
Subject: Re: High Sensitivity MS/MS Sequencing
Regarding Ken Williams questions about sequencing by MS/MS:
The best work in this area is being done by academic labs that are highly
specialized and have a uniquely important feature not available to core
labs or private industry: highly motivated and bright labor that is
cheap--graduate students. The investment of time and training necessary
to do a competent job in the area of true de novo peptide sequencing on
trace samples precludes most core labs from getting into it. Both Harvard
and the University of Virginia are exceptions: they maintain core labs
that do have this
capability, but I'm unsure how much gel work they are doing on a routine
basis. Some people to contact are Bill Lane (Harvard) and Nick Sherman or
Mike Kinter at the University of Virginia Medical Center. This is not to
say others are not also doing a good job--I just happen to know people in
these two core facilities.
One other comment: genes do not give complete covalent structure
information--as long as someone somewhere needs complete information on a
peptide or protein, protein microchemistry will have a place in the
universe! Mass spectrometers are somewhat unique in their ability to
define a complete structure, and are important tools for that reason. But
the don't work well for peptide sequencing in the hands of people that do
not have
excellent skills at the front end: making derivatives at trace levels,
cleanup, small scale separations, making the transblots, doing the in situ
digestions, etc. Having a mass spec that can see a few fmol of peptide
buys you nothing without these other important skills and a broad
knowledge of protein CHEMISTRY!
Murray Hackett
Department of Medicinal Chemistry
Box 357610
University of Washington
Seattle, Washington 98195
(206) 616-4586
fax (206) 685-3252
To follow up on Murray's comments our Mass Spec Sequencing Program is operating at a level approaching near capacity which is one reason we are expanding our instrumentation. Many of the projects brought to us simply require identification of a protein. The most typical sample source is a 1D or 2D gel band or spot which we process and generally confirm 10-70% of the total sequence, if the sequence is known. For sequences which are uncertain or not described in the various data bases we usual confirm our "calls" by performing ms/ms on synthetic peptides with sequences which are based on our deductions from the ms spectra.
The two key components to a successful ms lab is have an understanding of proteins, protein chemistry and the ability to handle low amounts of proteins and have talented, dedicated staff operating the mass spectrometers. It is for these reasons we set-up the mass spectrometery program within our existing Biomolecular Research Facility so that expertise in various areas could be called upon to solve the often complex and confusing problems brought to the facility by its users.
Jay Fox
Jay W. Fox,
Professor of Microbiology
Director; Biomolecular Research Facility
Phone: 804-924-0050
FAX: 804-982-2514