Computational protocol: Gene expression in the chicken caecum is dependent on microbiota composition

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Protocol publication

[…] Acetone precipitated protein pellets were dissolved in 300 μL of 8 M urea and processed following to the FASP protocol [] using 10 kDa MWCO Vivacon 500 filtration device (Sartorius Stedim Biotech). Initial protein washing was performed with 8 M urea followed by centrifugation for 12 min at 12 000 g. The reduction of disulfide bonds was performed with 10 mM dithiothreitol for 15 min at room temperature and acetylation was done with 50 mM iodoacetamide for 15 min at room temperature. After 3 washes with 10 mM triethylammonium bicarbonate, trypsin (Promega) was added at a 1:50 ratio and the digestion proceeded for 16 h at 30 °C.Proteins were quantified by 2 different protocols, either using stable isotope dimethyl labelling protocol or by label free analysis. When using dimethyl labelling protocol, samples were pooled and a single ratio of protein abundance in experimental and control group was obtained. On the other hand, label free analysis was performed for each sample individually thus allowing for statistical analysis.In stable isotope dimethyl labelling protocol analysis, total peptide concentration was determined spectrophotometrically (Nanodrop, Thermo Scientific) in each sample and samples from all germ-free and all conventional chickens in the experiment 1 were pooled. Pooled samples were then labelled using the stable isotope dimethyl labelling protocol [], mixed and 3 subfractions were prepared using Oasis MCX Extraction Cartridges (Waters). The subfractions were desalted on SPE C18 Extraction Cartridges (Empore) and concentrated in a SpeedVac (Thermo Scientific) prior to LC–MS/MS. This analysis was performed only for pooled samples from all germ-free and all conventional chickens collected in the experiment 1.LC–MS/MS analysis of both labelled and label-free tryptic peptides was performed using a Dionex UltiMate 3000 RSLC liquid chromatograph connected to a LTQ-Orbitrap Velos Pro hybrid mass spectrometer (Thermo Scientific). For each analysis, 5 μg of peptide sample was used. Each sample was separated on an EASY Spray C18 column (length 25 cm, I.D. 75 μm, particles 3 μm) using 300 nL/min flow rate of solvent A (0.1% formic acid) and solvent B [0.1% formic acid in 20/80 H2O/ACN (vol/vol)] and 150 min long reverse-phase gradient with concentration of solvent B gradually increasing from 4 to 40%. From MS spectra (Orbitrap analyzer, 30 000 FWHM resolution, mass range 390–1700 m/z), the 10 most intensive peptides were fragmented using CID fragmentation (normalized collision energy 35) followed by MS/MS scan (LTQ analyzer). Raw LC–MS/MS data were analyzed using Proteome Discoverer v1.4. MS/MS spectra identification was performed by the SEQUEST algorithm using chicken protein sequence database. Precursor and fragment mass tolerance were 10 ppm and 0.5 Da respectively. Carbamidomethylation (C) and oxidation (M) were set as static and dynamic modifications, respectively. Dimethylation (N-term and K) was set as static modification in the label-based analysis. Only peptides with a false discovery rate ≤ 5% were considered. […]

Pipeline specifications

Software tools Proteome Discoverer, Comet
Application MS-based untargeted proteomics
Organisms Gallus gallus, Escherichia coli, Enterococcus faecium
Chemicals Cystathionine