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I was totally lost when I tried to identify spot 23 in Figure 2. We understand this concern.
MA-plots are helpful to identify spot artifacts and to detect intensity-dependent patterns in the log ratios M. For the within-array normalization we used the robust scatter plot smoother loess (with the parameter span = 0.75) implemented in R statistical software [ 77] to perform a fit with a polynomial surface to the MA-plots of the raw data.
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The Otsu algorithm [ 56] was used to identify spots.
Then, a fluorescence intensity threshold was selected to identify spots within an expected range of sizes.
The resulting normalized spot volumes were subsequently analyzed in order to identify spots with different protein expression between groups.
The "SPOT" software package (http://www.experimental.act.cmis.csiro.au/Spot/index.php) was used to identify spots by adaptive segmentation method and subtract backgrounds utilizing morphological opening approach.
DeCyder BVA software was then used to match simultaneously all spot maps from 2D-DIGEs and to identify spots with statistical variations using the Student's t-test.
Normalized spot volumes between samples were compared to identify spots with significantly different volumes (P < 0.05; Student's t-test) between control and treated samples.
The 'SPOT' software package [ 44] was used to identify spots using the adaptive segmentation method and subtract backgrounds utilizing the morphological opening approach [ 45, 46].
The protein abundance is presented as the percentage of the total spot volume associated with each identified spot.
Second identified spot was oxygen-evolving enhancer protein 1 (OEE33, gene name psbO).
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