Mucus samples for two-dimensional electrophoresis (2-D PAGE) protocols were solubilized in equal volume of ice-cold lysis buffer (7 M urea; 2 M thiourea, 2% w/v CHAPS and 1% protease inhibitor mixture) and centrifuged at 20,000 g for 15 min at 4°C, whereas the resultant supernatant was aliquoted avoiding pellet resuspension and surface lipid layer. The supernatants obtained were submitted to a clean-up procedure (ReadyPrep 2-d clean-up kit, Bio-Rad) in order to enhance protein extraction as described in Sanahuja and Ibarz (44). The proteome map of soluble epidermal mucus proteins was obtained by 2D-electrophoresis. Protein concentration was determined by Bradford assay with bovine serum albumin as standard (Bio-Rad).

Pools of three mucus samples were prepared in order to obtain 450 µg of protein dissolved in 450 µL of rehydration buffer containing 7M urea, 2M thiourea, 2% w/v CHAPS, and 0.5% v/v IPG buffer, 80 mM DTT and 0.002% bromophenol blue. Five samples of skin mucus protein extract from each dietary condition (Control and SDPP diets) were loaded onto 24 cm, pH 3–10 NL IPG strips (GE Healthcare, Madrid, Spain). Isoelectric-focusing was performed using an IPGhor instrument (Amersham Biosciences), following the manufacturer’s instructions (active rehydratation at 50 V for 12 h followed by linear gradient from 500 to 8000 V until 48,000 V/h). The focused strips were equilibrated in two steps as follows: 15 min with equilibration buffer I (65 mM DTT, 50 mM Tris-HCl, 6 M urea, 30% glycerol, 2% SDS, bromophenol blue) and then, 15 min with equilibration buffer II (135 mM iodoacetamide, 50 mM Tris-HCl, 6 M urea, 30% glycerol, 2% SDS, bromophenol blue). Equilibrated strips were set directly onto 12.5% polyacrylamide gels, sealed with 0.5% w/v agarose, and separated at a constant voltage of 50 V for 30 min followed by 200 V for about 6 h, until the blue dye reached the bottom of an Ettan DALT II system (Ammersham Biosciences, Stockholm, Sweden). Proteins were fixed for 1 h in methanol: acetic acid 40:10 and stained overnight using colloidal Coomassie blue G-250. Gel staining was removed by consecutive washing steps with distilled water until the best visualization was achieved.

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).

Dried-down peptide mixtures were analyzed in a nanoAcquity liquid chromatographer (Waters) coupled to a LTQ-Orbitrap Velos (Thermo Scientific) mass spectrometer. Tryptic digests were resuspended in 1% FA solution and an aliquot was injected for chromatographic separation. Peptides were trapped on a Symmetry C18TM trap column (5 µm 180 µm x 20mm, Waters), and separated using a C18 reverse phase capillary column (ACQUITY UPLC M-Class Peptide BEH column; 130 Å, 1.7µm, 75 µm x 250mm, Waters). The gradient used for the elution of the peptides was 1 to 40% B in 20 min, followed by gradient from 40 to 60% during 5 min (A:0.1% FA; B: 100% CAN, 0.1% FA), with a flow rate of 250 nl/min. Eluted peptides were subjected to electrospray ionization in an emitter needle (PicoTipTM, New Objective) with an applied voltage of 2,000 V. Peptide masses (m/z 300–1,700) were analyzed in data dependent mode where a full Scan MS was acquired in the Orbitrap with a resolution of 60,000 FWHM at 400 m/z. Up to the 10th most abundant peptides (minimum intensity of 500 counts) were selected from each MS scan and then fragmented in the linear ion trap using CID (38% normalized collision energy) with helium as the collision gas. The scan time settings were: Full MS: 250 ms (1 microscan) and MSn: 120 ms. Generated.raw data files were collected with Thermo Xcalibur (v.2.2).

Files obtained from mass spectrometry analyses were used to search against the public database Uniprot Actinopterygii (v.23/3/17). A database containing common laboratory contaminant proteins was added to this database. The software used as Thermo Proteome Discoverer (v1.4.1.14) with Sequest HT as the search engine. The following search parameters were applied: two missed cleavage sites as well as fixed and variable modifications; carbamidomethyl of cysteine and oxidation of methionine, respectively. Peptide tolerance was 10 ppm and 0.6 Da for MS and MS/MS spectra, respectively. Both target and decoy databases were searched in order to obtain a false discovery rate (FDR), and thus, estimate the number of incorrect peptide-spectrum matches that exceeded a given threshold. The results were filtered so only proteins identified with at least two high confidence (FDR >1%) peptides were included in the lists. To sort the search results, proteins were ranked by a first criterion of the higher Score together with and a second criterion of the higher number of Sequence Coverage and Peptides matched. The principal component analysis (PCA) was used to check the quality of the data from each replicate and identify the subsets of samples that are associated with the two different groups under study. The protein intensity values (log2-expression ratios) were represented by a hierarchical clustering heatmap analysis using MeV software (v4.0), with Pearson distance and average linkage.

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