News Update on 2-D gel Capabilities for
Proteomics
August 2003
Several
improvements in the Hartwell Center 's 2-D gel electrophoresis service
for proteomic analysis have been implemented during the last three months. The
principal changes are as follows:
Two
staff members are now involved in the operation of the 2-D gel
service. The increased efficiency has resulted in a shortening
of the time taken for each project. Waiting times for initiation
of new projects have been correspondingly shortened.
Electrophoresis
systems from BioRad now allow twelve 10 cm and six 17 cm gels
to be run simultaneously. Taking into account the operations
involved in sample preparation, data analysis and protein identification,
the Hartwell Center can sustain production of 12 gels a week
on
an ongoing basis.
Sypro
Ruby dye from Molecular Probes is now routinely used for gel
staining. This provides high sensitivity, permitting protein
loads of
100-200 ug to yield information for up to 1,200 spots per gel,
depending on the nature of the lysate being analyzed.
A
new image analysis software package, Progenesis Workstation from
Nonlinear Corp. has been brought on-line. This provides a higher
degree of automation than our previous software, and greatly
reduces
the time needed for image analysis, a major rate-limiting factor
in throughput.
An
upgraded Model 4700 Proteomics Analyzer from Applied Biosystems,
a tandem TOF/TOF mass spectrometer, was installed in May, 2003
and has now been put into production. This instrument operates
at very high speed to produce peptide mass fingerprint and MS/MS
data for protein identification. Together with robotic systems
for spot picking and in-gel digestion, this instrument permits
large numbers of protein spots to be identified at one time.
It is therefore now possible to screen a substantial portion of
the spots on a gel. Charge-back fees have been reduced to $10.00
per protein spot.
For
database searching of mass spectra, use of GPS Explorer software
from Applied Biosystems has been tested. This system employs
the Mascot search routine from Matrix Science for identification
of proteins. With the use of this software, database search
times will shorten considerably. In cases where it is important
to check the output of the automated search engine manually,
as,
for example, when the identification of a critical protein rests
on the assignment of a single tryptic peptide, the amount of
time
taken to confirm results will be the rate-limiting factor in
throughput.
The
Hartwell Center has also developed methods for studying protein
expression that are complementary or alternative to 2-D gel electrophoresis. More details of these will be forthcoming shortly. Meanwhile,
we encourage investigators to make use of the enhanced capabilities
of the 2-D gel analysis service as the need arises.
