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iTRAQ labeling with two 8-plex iTRAQ kits was performed in the Proteomic facility of the Institute of Biomedicine of Seville using their standard protocols. Individual liver samples were isolated in urea lysis buffer (8 M urea, 25 mM Tris, 100 mM NaCl, 25 mM NaF, 10 mM Na4P2O7, 50 mM β-glycerophospate, 1 mM Na3VO4, 1:100 protease inhibitors and 1:100 deacetylase inhibitors, pH 8). Samples were sonicated for 10 seconds and centrifuged at 20000 x g for 15 min at 4 ºC. The supernatant was harvested and stored at -80º C. Then, samples were reduced with 50 mM tris-(2-carboxyethyl)phosphine (AB Sciex) at 60 °C for 1 hour with shaking and were subsequently alkylated using 200 mM S-methyl methanethiosulfonate (AB Sciex) for 30 min at room temperature. Samples were then trypsinized at 37 °C in a 10:1 ratio (w/w) of substrate/enzyme in a water bath overnight (Promega). Then, samples were speed-vac dried. The iTRAQ-labeling assay was conducted according to the manufacturer’s instructions (iTRAQ 8-plex, AB Sciex). Briefly, peptides were reconstituted in 1M triethylammonium bicarbonate (Sigma-Aldrich St. Louis, MO, USA), 0.05% SDS, 1:100 phosphatase inhibitor cocktail, 1:100 protease inhibitor cocktail, and 0.002% benzonase (Novagen, Argentina) and labeled with one isobaric amine-reactive tag. After 2 hoursof incubation, labeled samples were pooled, dried at 45 °C, and stored overnight at 4 °C. iTRAQ-labeled samples were desalted using Oasis HLB C18 cartridges (Waters, Milford, MA, USA) and dried using a vacuum centrifuge. Peptides were then prefractionated using MCX Oasis columns (Waters) and increasing concentrations (50-2000 mM) of ammonium formate. Fractions were collected, individually washed using C18 cartridges and dried. Peptides from each fraction were separated using nano-liquid chromatography (nano LC 1000, Thermo Scientific) and analyzed by means of nano-electrospray ionization (Proxeon Biosystems, Odense, Denmark) connected to a Q Exactive Plus Orbitrap mass spectrometer (Thermo Scientific). Briefly, 13 µL of each fraction was loaded, preconcentrated, and washed in an Acclaim PepMap (75 µm × 2 cm, nanoViper, C18, 3 µm, 100 Å) precolumn (Thermo Scientific). Peptides were separated in an analytical column (75 µm × 15 cm, nanoViper, C18, 2 µm, 100 Å, Acclaim PepMap RSLC) for 240 min at 200 nL/min (Thermo Scientific). Peptides were eluted with a gradient of buffer A (0.1% formic acid, 100% H2O) to buffer B (0.1% formic acid, 100% acetonitrile).Files contain data from the different fractions for iTRAQ experiments used for protein detection in RAW format. |
