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2D-ES-10: COMPUTERIZED COMPARISONS
OF 2D GEL PATTERNS FOR DIFFERENCES. This analysis includes laser
scanning and computerized analysis with SameSpots software from Non-Linear
Dynamics. Requires prior 2D electrophoresis but free duplicate gels are run
and analyzed to confirm differences for samples scheduled for computerized
comparisons. Generally 400-800 spots are quantified per Standard Format gel;
800-1200 spots are quantified per Large Format gel. Spot density values are
expressed as spot percentages (individual spot density as a percentage of
total density in all spots analyzed) to normalize for differences in sample
loading or staining. Results are presented in a complete report including a
summary table showing spot number, pI, MW, ratios (fold difference) and p
values as a second measure of difference. The final report also includes figures
of images showing numbering, montage images for every differing protein spot,
methods and pH gradient plot. A CD containing data and image files is included
in the package. Note that our reports focus on proteins
differences between samples. Picking protein spots later for identification
by mass spectrometry is straightforward. Just send us a list of spot numbers and
we'll take it from there.
Price: 2D-ES-10: Standard Format: duplicate gels
analyzed and averaged per sample. Per comparison, $450. Large Format:
2D-ES-10LF: duplicate gels analyzed and averaged per sample. Per comparison,
$500.
What is included in our 2D gel written
report and what is on the CD? We don’t include all the spot data in the
written reports because it makes them difficult to read. For example, spot
numbers become so numerous that
they obscure spot outlines on the figures. However, data for all polypeptide
spots analyzed, including unchanging ones, is always included in an Excel table
on the CD.
The standard written report consists of a cover page,
followed by a master table showing the spot #, isoelectric point, molecular
weight and spot % (spot density above background as a percent of all spots
measured) for changing protein spots along with the fold difference between the
samples, and p value for the difference. Anova values are included upon request.
The table is followed by figures of gel images showing the spot positions and
then a spot montage figure for each changing spot. The montage shows closeup
views of spot shape and outline for every gel in the set. In addition to clearly
showing significant changes between 2D gel patterns, our reports allow our Lab
Manager to easily find the proteins for subsequent spot cutting for mass
spectrometry. A CD containing: the report file, copies of the jpg images used
in the report, optional electronic photos (tif and jpg color images) as well as
an Excel file giving all spot data, accompanies each report.
How does SameSpots software work?
Comparison of Progenesis Discovery
and SameSpots (SS)
SS Cutoff for Differences between samples
How does SameSpots software work? The 2D gel images are first aligned to exactly match using an alignment module of the software. The Analyst then chooses an image as reference for automatic alignment and inserts localized seed matches at various points for each pair. The computer screen, shown below, provides four views of the zoomed matched area that help the analyst decide when the image alignments are perfect.
Figure 1. SameSpots computer screen during alignment of 2D gel images.
Next the aligned images are imported into the SameSpots module of the software where polypeptide spots are detected automatically on a single reference image and carefully hand corrected on one image. Identical spot outlines are then propagated to all the images. The Analyst goes through each image and checks to make sure the outlines are accurately placed. He/she goes back and realigns areas as necessary and redraws outlines if required. The alignment step is much less time-consuming than correcting spot outlines on every image. Every spot is identical on every gel facilitating statistical analyses such as Anova and Student’s t-test. Once the Analyst is satisfied, a report is generated and then checked by QA personnel before going out to the client.
The three examples are laid out to show spot outlines in montage view (top), plots of spot volume vs ug loaded (middle), and a bar graph showing spot percentage values for each gel (bottom). Results for clients are always reported in spot percentages (spot density taken as a percentage of the total density of all spots analyzed) because it corrects for any differences in loading and staining. Note that the montage images may look a little different between SS and PD because screen image contrast varied. The latter is a cosmetic adjustment that doesn't affect quantification.
Overall, the figures show that SS gives essentially the same results as PD. Note also that the middle plots of spot volume versus ug protein loaded give straight lines, confirming that the multi-step procedures of 2D electrophoresis and Coomassie blue staining give quantitative results.
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Figure 2: PD (blue) and SS (yellow) analysis results for a dark polypeptide spot of MW 60,800, pI 5.4 (#13). The top section shows spot outlines in montage view on 9 gels loaded with 200, 400 and 600 ug protein in triplicate. Screen image contrast varies between the montages but that doesn't affect results. The middle section shows plots of spot integrated density above background versus ug loaded on gel. The bottom section shows spot % (of total density of 60 spots) versus ug loaded on the gel. |
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Figure 3.
PD (blue) and SS (yellow) analysis results for a high molecular weight polypeptide spot
300,000, pI 5.7 (#1). The top section shows spot outlines in
montage view on 9 gels loaded with 200, 400 and 600 ug protein in
triplicate. The middle section shows plots of spot integrated density
above background versus ug loaded on gel. The bottom section shows spot % (of
total density of 60 spots) versus ug loaded on the gel. |
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Figure 4: PD (blue) and SS (yellow) analysis results for a faint polypeptide spot of MW 14,800, pI 5.7 (#57). The top section shows spot outlines in montage view on 9 gels loaded with 200, 400 and 600 ug protein in triplicate. The middle section shows plots of spot integrated density above background versus ug loaded on gel. The bottom section shows spot % (of total density of 60 spots) versus ug loaded on the gel. |
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SS Cutoff for Differences: To reiterate, for the new SS software spot outlines are automatically generated for all the images except the first, which is corrected by hand. Previously we hand-corrected all the images, which took a great deal of time. The new SS method is as reliable as the old, but has the consequence that when proteins disappear or have lower abundance in one sample, the outlines are drawn as large as the largest spot in the series on all the images. Since background subtraction is not perfect, a value always comes up for the low or missing spot. (For the old software the missing spot outline was omitted or drawn very small.) Thus spot ratios for changing proteins with the new software are smaller than they used to be; our old criteria for >3-fold differences is too high. Automatic statistical calculations are provided in SS and can be used to find legitimate differences between sets of gels. So we now use a double criteria for judging if protein spots are different: fold difference and p value. We check by montage view and report all spots that differ between the various groups with >1.7 fold change with a p-value of 0.05 or less. We also check any additional spots with a p value <0.0009 regardless of the fold change, and any spots with a 3-fold or grater ratio regardless of the p-value.
