Gel Image Analysis and Protein Digestion

Coomassie blue stained gels were scanned in a calibrated Imagescanner (Bio-Rad, Spain) and digital images captured using Quantity-One software (Bio-Rad). The images were saved as uncompressed TIFF files. Gel images were analyzed using the software package ImageMaster 2D, version 6.01 (GE Healthcare, Spain). Proteins were detected using the automated routine of ImageMaster 2.0 software, combined with manual editing when necessary to remove artefacts. The background was removed and normalized volumes were calculated as follows: the volume of each protein spot was divided by the total volume of all the protein spots included in the analysis. Normalized protein spot values were used to select the 300 most abundant proteins in each condition to be further analyzed for their differential expression. The obtained protein spots with differential expression, henceforth differential expressed spots (DESs) were manually cut from the gel and in-gel tryptic digestion was performed in an InvestigatorTM Progest (Genomic Solution) automatic protein digestion system as it was detailed for fish mucus samples in Sanahuja and Ibarz (44).