Our SDS-PAGE results support the 2DE findings of altered SAL concentrations; this protein was found as a main component of two bands with down-regulated intensities in GRR. applied for the search of proteins that could help understand disease mechanisms and be used for early disease detection. Two proteins were detected as possible markers of growth-rate retardation, specifically S100A12 and carbonic anhydrase VI. A decrease in innate immune response was confirmed in pigs with growth-rate retardation, however further studies should be necessary to understand the role of the different CA VI proteoforms observed. 0.05 with the statistical program GraphPad Prism 6 (Graph Pad Software Inc., La Jolla, CA, USA). 2.4. Two-Dimensional Gel Electrophoresis (2DE) To perform the protein profile Gemifloxacin (mesylate) comparison between control pigs Gemifloxacin (mesylate) and pigs with GRR, 2DE was used as previously described [16]. Briefly, 30 Gemifloxacin (mesylate) g of saliva proteins in rehydration buffer were rehydrated into immobilized pH gradient (IPG) strips with 11 cm long nonlinear gradients pH 3C11 (GE Healthcare Life Sciences, Munich, Germany) and subjected to isoelectric focusing in a Multiphor II electrophoresis chamber (GE Healthcare Life Sciences, Munich, Germany). For the second dimension, the IPG strips were equilibrated with a 2% DTT solution, followed with a 2.5% of iodoacetamide solution, and subjected to SDS-PAGE on homemade 10C15% polyacrylamide gradient gels of 140 140 1.5 mm in a vertical chamber (SE600 Chroma, Hoefer, INC., Holliston, MA, USA). Protein patterns were silver-stained according to general protocols [17] and scanned in an image scanner (ImageScanner II, GE Healthcare Life Sciences. Uppsala, Sweden). Images were evaluated for spot detection and matching using specific software (ImageMaster 2D Platinum 7.0, GE Healthcare Life Sciences, Uppsala, LDH-B antibody Sweden). The relative percentage of spot volumes from each group of animals was statistically compared using a using the following search parameters: global modification carbamidomethylation on cysteine; variable modifications oxidation on methionine; deamidation on asparagine and glutamine as well as formation of pyroglutamic acid; enzyme specificity trypsin; charge state z = 1; MS tolerance 100 ppm; MS/MS tolerance 1 Da; two missed cleavages allowed; significance threshold 0.05. 2.8. Protein Identification by Q Exactive HF Orbitrap Mass Spectrometry Dried peptides were resuspended in 0.1% TFA for the LC-MS/MS analysis and separated on a nano-HPLC Ultimate 3000 RSLC system (Dionex). Sample pre-concentration and desalting was accomplished with a 5 mm Acclaim PepMap -Precolumn (Dionex). For sample loading and desalting 2% ACN in ultra-pure H2O with 0.05% TFA was used as a mobile phase with a flow rate of 5 L/min. Separation of peptides was performed on a 25 cm Acclaim PepMap C18 column with a flow rate of 300 nL/min. The gradient started with 4% B (80% ACN with 0.08% formic acid) and increased to 9% B in 7 min, to 31% in 30 min and to 44% in additional 5 min. It was followed by a washing step with 95% B, Mobile Phase A consisted of ultra-pure H2O with 0.1% formic acid. For MS analysis, the LC was directly coupled to a high-resolution Q Exactive HF Orbitrap mass spectrometer. MS full scans were performed in the ultrahigh-field Orbitrap mass analyzer in ranges 350?2000 with a resolution of 60,000, the maximum injection time (MIT) was 50 ms and the automatic gain control (AGC) was set to 3e6. The top 10 intense ions were subjected to Orbitrap for further fragmentation via high energy collision dissociation (HCD) activation over a mass range between 200 and 2000 at a resolution of 15,000 with the intensity threshold at 4e3. Ions with charge state +1, +7, +8 and +8 were excluded. Normalized collision energy (NCE) was set at 28. For each scan. the AGC was set at 5e4 and the MIT was 50 ms. Dynamic exclusion of precursor ion masses over a time window of 30 s was used to suppress repeated peak fragmentation. Database search was performed using an in-house Mascot server (version 2.4.1., Matrix Science, Boston, MA) with following settings: The protein database consisted of amino acid sequences downloaded from UniProt for taxonomy 0.05. Finally, taking into account the identified proteins, function information was annotated from UniProt database [21]. 2.9. Protein Quantification by Western Blot One of the proteins found regulated in SDS-PAGE, S100A12, was subjected to validation analysis. Since the.
