Protein Extraction and Trypsin Digestion of L. paraplantarum RX-8

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).