Chicken deubiquitinases in spleen, cecum and liver
This study entails a chemical proteomic experiment performed to identify deubiquitinases in the chicken tissues, including spleen, cecum, and liver, based on the reactivity of these enzymes with active site-directed probes.
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Four male specimens of commercial broiler chickens at the end of their grow-out period (60-64 days) were used. After dissection, each tissue sample was placed in a sterile bag and transported on ice to a freezer. We used three biological replicates of cecum samples, and four replicates for liver and spleen samples.The tissue sections were homogenized by using the FastPrep-24 instrument (MPBio Inc.), where Matrix A beads were utilized and by using the cooling adapter filled with dry ice. Samples were homogenized by running the instrument 4 times for 30 seconds at 6.0 speed and letting samples cool on ice for 5 minutes between each run. To lyse the samples, samples were resuspended in lysis buffer containing 0.5% NP-40, 0.15 M NaCl, 0.02 M CaCl2·2H2O, 0.05 M Tris, pH 7.4 supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF). Samples were incubated on ice for 10 minutes and centrifuged at 14,000x g for 10 minutes at 4°C. The supernatant was removed to a new tube and filtered by using centrifugal filter units (Ultrafree-MC 0.1 µm units; Millipore, USA). Protein was quantified by the DC Protein Assay Kit according to the manufacturer’s instructions (Bio-Rad, USA) and 10 mg per sample was used for subsequent steps. DUB labeling in chicken tissues The DUBs were labeled in tissue lysates by using active site-targeted ubiquitin-specific activity probes as we have previously described (23). Prior to labeling, we diluted the samples to 5 mg/ml, and incubated cells with DTT [1 mM] for 10 minutes on ice. Ub-VS-HA (Boston Biochem Inc) probe was then added to each sample and incubate for 30 minutes at 37°C. After labeling was complete, a small fraction (10%) of the sample was removed for western blotting analysis, and otherwise, the samples were put on ice and mixed with 100 µl of equilibrated EZview anti-HA beads (80 ul/sample). Samples were then incubated on ice with rocking for 2 hours, followed by washes (4 x 800 µl) by using the lysis buffer and finally 150mM NaCl, 50 mM Tris pH 7.5. Proteins bound to the anti-HA resin were eluted by boiling in 0.1% RapiGest SF (Waters Inc.) buffer at 95°C for 5 min. The eluates were precipitated by 0.18 volumes of 100% TCA for 20 min on ice and spun for 30 minutes at 21,000 x g. The supernatant was then removed and precipitated washed with 500 µl of -20°C acetone. Samples were spin, acetone removed, and the precipitate was left to air dry. Samples were then analyzed by Ultrahigh-Performance Liquid Chromatography (UHPLC) coupled to Orbitrap Fusion mass spectrometer (MS) (Thermo Scientific).