Computational protocol: Secretome profiling of Propionibacterium freudenreichii reveals highly variable responses even among the closely related strains

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Protocol publication

[…] Four biological replica cultures of JS14 and JS22 (45 ml each) were centrifuged (5000 g, 15 min, 4°C) to collect supernatants. The supernatants were supplemented with protease inhibitors as described above, and the proteins were precipitated by the addition of 10% (w/v) TCA. After overnight incubation at 4°C, the proteins were pelleted by centrifugation (8000 g, 45 min and 4°C). The protein pellets were washed once with 96% ice‐cold EtOH. After centrifugation (8000 g, 10 min, 4°C), the air‐dried protein pellets were solubilized in 150 μl 100 mM Tris–HCl buffer (pH 8.0) containing 2% SDS (w/v). Solubilized proteins were subjected to an additional purification step using the 2D‐Clean‐up Kit (GE Healthcare) following the manufacturer's instructions. The precipitated proteins were finally solubilized in 100 μl of UTCT (7 M urea, 2 M thiourea, 4% CHAPS and 30 mM Trisma Base), and the protein concentrations were determined using a 2‐D Quant Kit (GE Healthcare). Equal amounts of solubilized proteins (100 μg) were separated using isoelectric focusing (IEF) with IPG strips (11 cm, pH 3–10/linear; GE Healthcare) that had been rehydrated overnight in De‐Streak solution supplemented with 1% IPG buffer pH 3–10 (GE Healthcare). The samples were applied using loading cups placed at the anode end of the strips, and the IEF was conducted at 20°C with the following parameters: 500 V for 500 Vh, linear ramping to 1000 V V for 800 Vh, linear ramping to 6000 V for 8800 Vh and holding at 6000 V for 2900 Vh. The current limit was set to 50 μA per strip. After IEF and the strip equilibration in buffers containing 50 mM Tris–HCl at pH 6.8, 6 M urea, 2% SDS, 20% glycerol, and alternatively either 2% DTT or 2.5% iodoacetamide (15 min each), the strips were loaded onto 12.5% Tris–HCl Criterion Precast Gels (Bio‐Rad), and the second‐dimensional protein separation was conducted using the Criterion Dodeca Cell (Bio‐Rad) in 1× Tris–Glycine–SDS buffer at 220 V at 16°C.After electrophoresis, the gels were stained with Sypro Orange (1:5000) using the previously described method (Malone et al., ) with the following modifications: the gels were fixed in a solution (100 ml) composed of 40% ethanol, 2% acetic acid and 0.0005% SDS for 1 h. The pre‐stain washing step was omitted, and two post‐staining rinses with fresh MilliQ water were applied to wash the gel prior to the acquisition of the gel images as described above. The 2‐DE images were analysed using the samespots software v4.5 (Totallab, UK) with four individual gel replicates from both strains. Non‐spot data from the 2‐DE images were first removed by cropping the images, which was followed by automatic normalization and selection of the reference image. Spot matching between different 2‐DE gels was manually improved by adding appropriate number of alignment vectors to each gel image. Spot volumes for all matched proteins within each analysis were calculated and normalized to the total spot volume to determine the average spot volume changes across all four replicates within both test groups. All spots showing a fold change ≥ 2.0 and P < 0.05 were selected for identification using in‐gel tryptic digestion coupled with LC‐MS/MS analysis.Each fluorescent 2‐DE gel was post‐stained with silver as described above. Selected protein spots were cut out of the gels and treated with trypsin, and the tryptic peptides were analysed by LC‐MS/MS as previously described (Koskenniemi et al., ). The MS/MS data were searched using the local mascot version 2.2 (Matrix Science, London, UK) against the in‐house database consisting of all predicted protein sequences deduced from the JS14 and JS22 genomes through the ProteinPilot interface. The search criteria for the Mascot searches were trypsin digestion with one missed cleavage allowed, carbamidomethyl modification of cysteine as a fixed modification, and oxidation of methionine as a variable modification. For the MS/MS spectra, both the maximum precursor ion mass tolerance and the MS/MS fragment ion mass tolerance were 50 ppm, and peptide charge states of +1, +2, +3 were used. A successful identification was reported when a Mascot identification score > 50 (P < 0.05) with a minimum of two identified peptides (ion score > 40, P < 0.05) was obtained. [...] Three biological replica cultures of JS14 and JS22 (30 ml each) were centrifuged (5000 g, 15 min, 4°C) to collect cells. Cells were washed twice with 100 mM sodium phosphate buffer (pH 6.8) and then suspended gently in 1× Laemmli buffer (Laemmli, ). Cells in Laemmli buffer were incubated on ice for 5 min to release non‐covalently bound proteins (Sleytr et al., ). Intact cells were removed by centrifugation (4000 g, 5 min, 4°C), and the recovered supernatants were incubated at 100°C for 10 min. Then, the denatured proteins were separated in precast 12.5% TGX gels as described above and the gels were stained with Coomassie Blue (R‐250) (Sigma‐Aldrich) for 1 h and destained by 30% MeOH three times (30 min each). The Coomassie Blue‐stained images were captured using the Alpha Imager HP. Selected protein bands were subjected to in‐gel tryptic digestion and LC‐MS/MS as previously described (Savijoki et al., ). The MS/MS data were searched using the Mascot search engine, and the search parameters and the in‐house protein databases created for JS14 and JS22 as described above. […]

Pipeline specifications

Software tools SameSpots, ProteinPilot, Mascot Server
Application MS-based untargeted proteomics
Organisms Propionibacterium freudenreichii, Bacteria
Chemicals Sodium Chloride