Protein concentration was measured by Bradford assay and 15 µg of protein solution was mixed with Laemlli buffer (62.5 mM Tris–HCl (pH 6.8), 10% glycerol, 1% SDS, 5% β-mercaptoethanol and 0.001% bromophenol blue). After boiling for 10 min, the reduced sample was loaded on a 1D SDS-PAGE gel. The molecular weight (MW) marker (Precision Plus Protein Standards, Bio-Rad, Nazareth Eke, Belgium) was loaded as reference to cut the lanes in five bands. The proteins were electromobilised at 150 V, the gel was then fixed and stained overnight with Coomassie brilliant blue G-250. After destaining in 30% methanol to obtain a clear background, the reduced and alkylated gel was sliced into five bands per lane. Each band was in-gel digested with trypsin and analysed via a fully automated LC MS/MS setup [19]. Briefly, 4 µL of a peptide solution was initially concentrated and desalted on a Zorbax 300SB-C18 trapping column (5 mm × 0.3 mm, Agilent, Santa Clara, CA, USA) at a 4 µL/min flow rate using a 2% (v/v) acetonitrile, 0.1% formic acid in water. After valve switching, the peptides were injected to and separated on a Zorbax 300SB-C18 analytical column, 150 mm × 75 µm (Agilent, Santa Clara, CA, USA), by a 30 min linear gradient ranging from 2% (v/v) to 50% (v/v) acetonitrile, 0.1% formic acid in water at a 300 nL/min flow rate. The eluting peptides were measured online on a LTQ-FT Ultra mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The FT-ICR mass analyser acquired MS scans at a resolution of 100 000 during the LC separation. The five most abundant molecular ions from each MS scan were automatically selected by the LTQ ion trap mass analyser. Subsequently, the precursor ions (charge state + 1 is rejected) were fragmented by collision-induced dissociation (CID), using normalised collision energy of 35.0%. After two occurrences, the precursor masses were excluded for 90 s.
Protein identification
Peak detection from raw data was performed by ExtractMSn version 1.0.0.8 (Thermo, default parameters), which determines the monoisotopic m/z ratio value and charged status of the precursor ions along with the m/z values and the intensities of the fragment ions. Mascot Daemon (Matrix Science, London, UK; version 2.4.1) was used to search the retrieved peak list against the NCBI non-redundant protein database. Search parameters were assuming the digestion enzyme trypsin with a fragment ion mass tolerance of 0.3 Da, and a parent ion tolerance of 10 ppm. Oxidation of methionine and carbamidomethylation of cysteine were specified as variable modifications.
The resulting Mascot DAT-files were loaded in Scaffold (Proteome Software Inc., Portland, OR, version 4.4.1) and the MS/MS data were analysed using X! Tandem (The GPM, thegpm.org; version CYCLONE 2010.12.01.1). X! Tandem was set up to search a subset of the reverse concatenated database using the same parameters as Mascot, but glutamate and glutamine to PyroGlu and ammonia-loss of the N-terminus and oxidation of methionine and carbamidomethylation of cysteine were specified as variable modifications. Scaffold was used to validate MS/MS based peptide and protein identifications.
The peptide identifications were accepted if they could be established at higher than 99.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.9% probability and contained at least two identified peptides. Protein probabilities were assigned by the ProteinProphet algorithm [20]. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into protein clusters. Quantification was based on exclusive spectral counting, a label free proteomics approach were the frequency of collecting a peptide precursor ion in MS/MS is used as a quantitative measure [21]. Spectral counts were normalized to the total number of spectra in the corresponding LC–MS run.
Ovotransferrin detection by enzyme-linked immunosorbent assay (ELISA)
In order to confirm the ovotransferrin results obtained from the proteomic analysis of faecal samples from the necrotic enteritis in vivo trials, faecal samples from another, independent, necrotic enteritis in vivo trial were used. Eight samples from the non-challenged control birds (receiving all predisposing factors, but not challenged with C. perfringens) and eight samples per necrosis score group (either mild, moderate or severe NE) from challenged birds were selected. Also litter samples collected at the day of necropsy were included (one litter sample per pen, with in total three samples from pens with non-challenged birds and three samples from pens with challenged birds). Additionally, the ovotransferrin concentration was determined in both faecal samples and litter samples derived from the coccidiosis trial. Therefore, 5 litter samples from pens with non-challenged birds and 6 litter samples from pens with Eimeria-challenged birds were used. Additionally, 20 faecal samples from either Eimeria-challenged birds (n = 10, one sample from each group) or their non-challenged controls (n = 10, one sample from each group) were selected.
Unprocessed faeces or homogenized litter material were thawed at room temperature. The faeces or litter material (150 mg) was diluted in 1500 µL TBS (50 mM Tris, 150 mM NaCl, pH = 7.2) with protease inhibitor cocktail (P2714, Sigma-Aldrich). The samples were mixed by vortex (2 × 1 min). Proteins (supernatants) were collected after centrifugation (13 000 × g, 10 min, 4 °C) and were used in duplicate (1/50 dilution) in the ELISA (Chicken Ovotransferrin ELISA, KT-530, Kamiya Biomedical Company, Tukwila, USA). The ELISA was performed according to the instructions of the manufacturer.
Ovotransferrin stability in faecal material
To assess the stability of ovotransferrin in chicken faeces, ovotransferrin (Conalbumin, Sigma-Aldrich) was spiked in two faecal samples from different birds to obtain a final concentration of 200 µg ovotransferrin per gram faeces. The spiked samples were aliquoted and incubated at room temperature for 0, 1, 6 or 24 h, proteins were extracted as described above and used in duplicate (1/50 dilution) on the chicken Ovotransferrin ELISA.
In order to elucidate whether the observed decline in ovotransferrin signal is due to proteolysis caused by proteases present in the faeces, a second spiking experiment was set up. Ovotransferrin was spiked in two faecal samples from different birds. However, before spiking a protease inhibitor cocktail (Sigma-Aldrich, P2714; inhibits serine, cysteine and metallo-proteases) was either or not added to the ovotransferrin. The samples were incubated for 0, 2 or 24 h at room temperature. Protein extraction and ELISA were performed as described above and the pH was measured after different incubation periods. The % recovery of ovotransferrin at each timepoint was calculated relative to the 0 h timepoint.
Statistical analysis
Normality of the data was tested with the D’Agostino-Pearson normality test. For the proteomic analysis of samples from the NE in vivo trials, a Fisher’s exact test was performed on the total spectral counts of the samples from non-challenged birds or the samples from birds with lesion score 3–4. Hochberg–Benjamini correction was performed to correct for multiple testing. Differences in ovotransferrin levels between necrotic enteritis severity groups (as measured by ELISA) were calculated using an a Kruskal–Wallis test, followed by Dunn’s post hoc test. Differences in ovotransferrin levels between the Eimeria-challenged and the non-challenged control group were calculated using a Mann–Whitney test. The Spearman rank correlation was used to assess the relationship between the ovotransferrin concentration in the faecal samples and either the necrotic enteritis lesion score or the coccidiosis score. Results were reported as means and standard error of the means (SEM).