Hello Kim,
For what its worth here are my thoughts.
I recently started a proteomics and metabolomics research group at a small
private research foundation. We are not necessarily a fee-for-service lab
but we have faced many of the same challenges. Our goals include an
ambitious plant proteomic effort.
1) pieced together
Our gel systems, picker/digester, and mass spec equipment were purchased
from 3 different manufacturers. We have purchased automation equipment (spot
picker, autodigester, MALDI spotter) but it still takes us 3 days extract,
rehydrate IEF, focus, run gel, and stain. With our current equipment and
staff, running parallel gels, we could easily run approx 12-24 gels per
week. (We use hand cast gels and allow for overnight polymerization). This
number could be doubled with more gel rigs. The real time consuming process
start after you have image the gels. Comparative gel analysis using
commercial software is still time an operator intensive (easily a day per
experiment). The a day to pick, start digestions, recovery peptides. The
amount of time varies for mass spec analysis and database searching but it
certainly is the rate limiting step. Saying that it takes us at least
another 3-5 days to mass map the digests and search the databases manually
for a single gel. Our program is further complicated by the lack of
relevant plant proteins in the database (even in light of the arabidopsis
effort). We often search the EST data bases as well making the searches
even longer. We are considering PE Biosystems automated MALDI spectral
acquisition and database searching software (AutoMS Fit). It looks very
promising. Bottom line, high throughput for us is approximately 25-50
proteins identified per week for two lab staff. I am very interested in the
numbers from other labs.
2)yes - not enough room to detail all.
3)both
We do mid sized to large gels. The mini gels are a good preview tool but
the larger gels allow greater protein loads and improved spatial resolutions
more amenable to spot picking. We currently use a 16x16cm setup and are
very happy with it. If you are working with limited protein sets then the
mini gels are usually acceptable. Larger gel options are available (20x20
or 24x24) and we see an approx 10% increase in the overall number of
detectable sample spots as we step up in size from the 16x16 to the 20x20,
however larger gels are increasingly more difficult to handle without
tearing and require more stain...$$$$$$ if you are using sypro ruby.
4)n/a
5)internal and pending NSF Plant genome $$$
6) Two - We currently have 1 master level person for gels, 1 PHD for mass
spec. We also have a B.S level person for metabolomics. We are way under
staffed. We currently have two immediate openings and find it difficult to
recruit the folks that we need given our location and current market
conditions. Our five year plan will look to fill 10 positions.
Besides good people, I believe the critical components center around a good
imager and software. A 16 bit system has the widest dynamic range but we
currently use a 12 bit system. The next critical component is the
comparative analysis software. You have many options Phoretics/Nonlinear,
BioRad, Compugen, Melanie..etc.
Staining - we use coomassie. We work with plants and tissue quantity is
usually not a limiting factor, thus we grind more to get more protein. My
minimal experience with fluorescent stains suggest that the 10x cost of
fluorescent stains over silver is not worth it, thus we use silver when
working at low levels. However, the concept of using multiple fluorescent
stains to tag multiple/different cell states which are then run on the same
gel does look very promising.
The final thought is that all 2D gel technologies may be surpassed by the
rapidly maturing ICAT, two dimensional LC/MS/MS approach to proteomics.
For more information on specific equipment, feel free to call.
Good Luck,
Lloyd
Lloyd W. Sumner, Ph.D.
Asst. Scientist & Head, Biological Mass Spectrometry
The Samuel Roberts Noble Foundation
PO Box 2180
2510 Sam Noble Parkway
Ardmore, OK 73402
Voice 580-221-7392
Fax 580-221-7380
Email lwsumner@Noble.Org <mailto:lwsumner@Noble.Org>
http://www.noble.org/Plantbio/MS/
-----Original Message-----
From: Helen Kim, Ph.D. [mailto:Helen.Kim@ccc.uab.edu]
Sent: Thursday, March 01, 2001 4:06 PM
To: Recipients of ABRF List
Subject: ATTN: directors of academic proteomics facilities
Hi everyone,
I'm totally new to ABRF; I went to San Diego, now know about 3 of you, but
don't who are directing proteomics facilties;
There were no formats at the meeting where the issue of running proteomics
facilities, versus doing your own, was addressed. Maybe it's too new, or
maybe there aren't enough academic facilities out there, or all the other
facilities are up and running, and don't need any further information.
Whatever the case, I'm wondering if those of you who are running
Proteomics facilities could share some information with me, and others who
may be interested:
1. did you buy an integrated system for analyzing your 2-D gels and mass
spectrometry, or piece it together? if you have an integrated system, which
system, and do you like it?
In particular, are you "high-throughput" i.e. automated?
2. if you had to do it over, would you buy a different system, or in any
way set up the lab differently?
3. What do you offer: 2-D gels, mini's versus large formats?
4. big question: how do you charge? by the gel? by the hour?
5. even bigger question: where did you get your funding???
6. besides youself (director), what personnel do you have,
someone dedicated to the gels?
someone dedicated to the Mass spec?
I am the director of the UAB 2-D Proteomics Laboratory, which is part of the
Comprehensive Cancer Center Mass Spectrometry Shared Facility which has
existed since 1993 (under Steve Barnes' direction). We started running
mini 2-D gels for a handful of investigators last summer, following up with
maldi-tof. Last month I got internal funding to establish the Proteomics
Lab as a UAB facility, and to "enhance" the capabilties of the Lab. But, of
course, the funds are not unlimited. At San Diego, it really wasn't helpful
getting sales pitches, they all say they have the best.
We have the BioRad non-UV scanner, and are using PDQuest. We are going to
definitely shift over to fluorescent dyes, and therefore need the higher
level scanner. We are on the verge of having to shift to some level of
automation, because user interest is high. Are the gel spotpickers and
automated il-gel digesters worth the money?
Also, is anyone processing samples before the gels, for the users? i.e. if
they say they think their protein of interest is in the nucleus, are you
fractionating their lysates for them, or is everyone keeping their sanity by
letting/making the user do all the sample preparation prior to the
electrophoresis?
That's probably enough for now. If you only feel like answering part of
the above, that's fine. Thanks in advance to those who do respond. Helen
Kim
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