Scan vac maxi beta

The liver samples were separately treated with liquid nitrogen and suspended in lysis buffer [8 M urea, 50 mM Tris 8.0, 1% NP40, 1% NaDOC (sodium deoxycholate), 5 mM dithiothreitol, 2 mM EDTA, 30 mM nicotinamide, 3 µm trichostatin A, 1% cocktail protease (Sigma, P8215, for use with fungal extracts) and 1% phosphatase inhibitor cocktail (Solarbio, P1260)]. The samples were sonicated and centrifuged at 4 °C for 10 min at 20,000 g to remove tissue fragments. The protein content was detected using a 2D Quant kit (GE Healthcare Life Sciences, PA, USA). Equal amounts of protein were reduced with 5 mM dithiothreitol for 45 min at 30 °C, alkylated with 30 mM iodoacetamide for 1 h in the dark at room temperature, and then precipitated with acetone on ice. The precipitate was washed with acetone and suspended in 0.1 M triethylammonium bicarbonate (TEAB) and digested with trypsin (Promega, Madison, WI) overnight at 37 °C. Digestion was stopped with 1% trifluoroacetic acid. The peptide obtained was cleaned using a Strata X C18 SPE column (Phenomenex, Torrance, CA, USA) and vacuum-dried in a Scan-Vac maxi-beta (Labogene, Alleroed, Denmark).

Cell samples of L. paraplantarum RX-8 in co-culture and mono-culture were collected at 24 h according to Section 2.5.1. The protein was extracted by using a lysis buffer (8 M urea, 50 mM Tris8.0, 1% NP40, 1% sodium deoxycholate, 5 mM dithiothreitol (DTT), 2 mM EDTA, 30 mM nicotinamide, and 3 μm trichostatin A), and, after sonication on ice, the total protein concentration of the supernatant, which was obtained by centrifugation (20,000 rpm, 10 min, 4°C), was determined by using a BCA Protein Assay kit. The protein sample was reduced by DTT (5 mM, 45 min, 30°C), later alkylated with 30 mM iodoacetamide (30 mM, 1 h, RT) in darkness, and then precipitated with ice-cold acetone. After being washed thrice with acetone, the precipitate was suspended in 0.1 M triethylammonium bicarbonate (TEAB) and digested with trypsin (1/25 protein mass, Promega) for 12 h at 37°C. Finally, the reaction was ended with 1% trifluoroacetic acid (TFA), and the resulting peptide was desalted with Strata X C18 SPE column (Phenomenex, Torrance, CA, USA) and vacuum-dried in Scanvac maxi-beta (Labogene, Alleroed, Denmark).