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The spots in the EBPR matchset were ranked according to their spot quantity as determined by PDQuest analysis.
The individual protein spot quantity was normalized as follows: the raw quantity of each spot in a member gel was divided by the total quantity of the valid spots in the gel, and normalized spot intensities were expressed in ppm.
The spot quantity table containing all matched spots was generated.
Spot quantity between SScFib+ and SScFib- patients did not differ significantly (Mann–Whitney U test, p > 0.05).
The normalized spot quantity was expressed as percentages of volume contributions (vol%) to facilitate the data compilation.
If co-migrations and multiple identifications were found in one spot, then peptides arbitrarily shared the spot quantity.
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In order to accurately compare spot quantities of the two samples, the volume of each protein spot was normalized and automatically calculated by the software as a single spot volume.
All normalized spot quantities from the gels were collectively analyzed as two independent groups "Tumor" and "Benign", which enables matching of multiple gel images from different patients to provide statistical data on average abundance for each protein spot among the DIGE gels included in analysis.
The spot quantities are then calculated as usual by summing the pixel intensities within the spot boundaries.
For comparison of spot quantities, spot volumes were normalized as a relative volume (% vol, a percentage of the total volumes of all of the spots in the gel).
The differences between the two study groups were then confirmed by analysing the spot quantities observed in single patients.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com