Date: Thu, 02 Jan 1997 18:05:00 -0500 (est)
From: "Williams, Ken" <WilliamsKR@MASPO1.MAS.YALE.EDU>
Subject: High Sensitivity MS/MS Sequencing
To: Recipients of ABRF List <abrf@aecom.yu.edu>
We are coming under ever increasing pressure to offer MS/MS sequencing
on very low/sub-pmol amounts of "unknown" proteins separated by SDS PAGE -
with the driving force being recent publications where this has been
accomplished in a manner that, according to some of our users is (and I
quote) "straightforward and relatively easy". Frankly, I could use some
help defending our unit and would very much appreciate any input along the
lines suggested by the following questions (brought to mind by the recent
note by David Andrews):
1. If anyone reading this message offers this service, I would very much
like to know if it is collaborative/non-collaborative, what kind of results
can routinely be obtained on lots of unknown samples, what are the service
charges/costs (by hour or sample, etc), what is the success rate with
differing amounts of sample, what equipment is being used, how much
experience is needed to accurately interpret the data , etc, etc.
2. Is there a review article somewhere that covers this topic - with
emphasis on amounts of protein that are generally needed to succeed.
3. Besides the Nature 379 (1996) p 466 and Anal. Chem 68 (1996) p 850
references - are there other references that clearly indicate the total
amount of SDS gel separated protein needed for internal MS/MS sequencing?
4. Since the human and several other genome projects will probably be
completed by the year 2005 - it seems to me this is a relatively "temporary"
problem in that within a few years the fraction of proteins submitted for
internal sequencing that eventually prove to be known (currently the
fraction is at 70% in our unit and rising about 10%/year) will be close to
100% - which will mean we will then need to identify proteins at very high
sensitivity but not necessarily carry out de novo MS/MS sequencing - which
seems to require much more expertise. Do you agree/disagree with this
assessment?
Very best wishes for the New Year and thanks very much for the help
that I know will soon be forthcoming,
Ken Williams