Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 35)

PDQuest ™ 2-D Analysis Software EXQuest Spot Cutter
Bio-Rad’s PDQuest 2-D analysis software is used
for analyzing and creating databases for 2-D
electrophoresis gels. It provides a series of “wizards”
for the analysis of digitized gel images and for spot
detection and quantitation, gel comparison, and
statistical analysis. The Experiment Wizard guides
selection of gels for analysis, detection of spots
of interest, creation of an experiment, and matching
of gels. The Spot Detection Wizard then guides the
identification and quantitation of the
spots in gel images.
After detection, gels in the same series are placed
in an experiment for comparison, statistical
analysis, and databasing. Histograms allow quick
comparisons of the quantities of the same spot
in all the gels in an experiment. Spots can also
be compared qualitatively, organized into user-
defined sets for further analysis, and annotated
and databased for easy identification. Spots from
Bio-Rad’s EXQuest spot cutter accurately locates
and excises protein bands or spots from gels
or blots and loads them into 96- or 384-well
microplates or 96-tube racks for downstream
processing and analysis. Its camera works with
PDQuest 2-D analysis software to visualize gels
and blots that are either visibly or fluorescently
stained. In 2-D electrophoresis applications,
PDQuest software tracks the protein bands
or spots through spot cutting and protein
identification, which is usually accomplished
using mass spectrometry.
The EXQuest spot cutter allows use of any common
proteome separation and staining methods:
■ ■
Freestanding or plastic- or glass-backed 2-D
and 1-D SDS-PAGE gels
■ ■
PVDF and nitrocellulose membrane blots
■ ■
Gels or membranes stained for proteins with
visible stains (such as silver and Coomassie blue
stains) or fluorescent stains (such as Flamingo
™ ,
Oriole ™ , and SYPRO Ruby protein stains)
ExQuest Spot Cutter
different experimental series can be organized and
compared in higher-level experiments. PDQuest can
be used to simultaneously analyze thousands of
spots on hundreds of gels. Data can be exported
to other applications, such as spreadsheets, for
further analysis.
PDQuest software has the further advantage of
integration with Bio-Rad’s EXQuest
™ spot cutter,
which accurately locates and excises protein
spots from 2-D gels or blots at high speed
(up to 600 spots per hour) and then loads them
into 96- or 384-well microplates or 96-tube
racks for downstream processing and analysis.
PDQuest has no imaging functions besides driving
the camera system in the ExQuest spot cutter, but it
can read and import multiple file formats from other
gel imaging software packages like Quantity One
® .
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2-D Electrophoresis Guide Theory and Product Selection Chapter 6: Image Acquisition, Analysis, and Spot Cutting
Many software packages for automatic gel comparison
are created with the assumption that the relative
positions of spots are altered only slightly relative to
each other, and they allocate the spots on this basis.
Prior to automatic gel comparison, PDQuest software
selects the best 2-D gel of a gel series as a reference,
or standard gel, and compares all other 2-D gels to
this gel. Proteins in a gel series that are not present in
the reference gel can be added automatically so that
the reference gel includes all proteins of a gel series.
PDQuest includes the ability to match spots with no
manual assistance, and it is possible to display up
to 100 enlarged details of 2-D gels on the screen
simultaneously, enabling rapid and error-free
determination of the matching quality.
Data Normalization
When comparing gels in an experiment, there is
often some variation in spot size and intensity among
gels that is not due to differential protein expression.
Multiple normalization methods can be used to
compensate for gel-to-gel variations in spot intensity
caused by inconsistencies in sample loading, gel
staining, and imaging. To accurately compare spot
quantities among gels, compensation for these
variations in spot intensity, which are not related to
expression levels, is required.
Image Optimization, Spot Detection, and Quantitation
Before any software can detect the protein spots of a
2-D gel, raw image data must be optimized and the
gel background subtracted.
PDQuest software models protein spots
mathematically as 3-D Gaussian distributions and
uses the models to determine protein maxima.
A 3-D Gaussian spot is a precise representation of an
original scanned spot. Gaussian curves are fitted to
the scanned spot in the X and Y dimensions, and then
additional modeling is performed to create the final
Gaussian spot. Using Gaussian modeling, it is possible
to accurately quantitate overlapping spots, spots in
gel streaks, and multiple spots in dense clusters.
The accuracy of automatic spot detection depends
on the quality of the 2-D gels and their images.
Correction capabilities of PDQuest software can be
used to add undetected spots to the list of spots or
to delete spots that arise from gel artifacts.
Gel Comparison
The next step in 2-D gel evaluation is identification
of proteins that are present in all gels of a series.
Since inherent problems with gel-to-gel reproducibility
affect the positions of spots within a gel series,
gel analysis software must be able to detect minor
shifts in individual spot position within the gel series.
Data Analysis and Reporting
With PDQuest software, all gels in a single experiment
are viewed as a unit. To compare gels from different
experiments, the reference images are compared.
In such comparisons, each spot is automatically
assigned a number such that identical spots have
identical numbers. In an experiment, the molecular
weight and pI values for known protein spots can
also be entered. With these data, PDQuest can
estimate molecular weight and pI values for all the
spots in the experiment.
Analysis sets allow the study of sets of proteins that
are statistically and scientifically significant and to
identify spots to cut using the ExQuest spot cutter.
There are six different kinds of analysis sets:
■ ■
Qualitative analysis sets — spots that are present
in one gel but not in another
■ ■
Quantitative analysis sets — spots whose intensity
(amount) has increased or decreased by a certain
degree, or whose intensity has changed above,
below, or within the fold change factor that you specify
■ ■
Statistical analysis sets — spots that are significant
according to the statistical test that you apply
■ ■
Arbitrary analysis sets — manually selected spots
■ ■
Boolean analysis sets — created by comparing
two or more analysis sets (for example, set C could
be made up of those spots present in both sets
A and B)
■ ■
Matching analysis sets — spots that are either
unique to one member or present in all members
Once proteins of interest are determined, the
corresponding analysis sets are uploaded to the spot
cutter. The spots of interest are then excised from the
gels and digested to release peptides for analysis by
various mass spectrometry methods.
Spot Cutting from 2-D Gels
Spots of interest can be excised from gels either
manually (for example, with a scalpel, razor blade,
or modified pipet tip) or with an automated spot
cutting system. The advantages of automated systems
are numerous and include improved precision and
reproducibility, tracking of gel spots, and decreased
risk of contamination. The excised gel plugs are
then transferred to microplates or other vessels for
digestion and further analysis.