The solution was mixed with an equal volume of 0.5-mm glass beads (Tomy Seiko, Tokyo, Japan). The cells were then disrupted mechanically
in triplicate by using Gamma-secretase inhibitor BeadSmash 12 (Wakenyaku, Kyoto, Japan) at 4°C, 4,000 × g for 1 min. The solution was centrifuged at 14,000 × g for 10 min, and the supernatant was collected. The supernatant was filtered by 0.45 μm Ultrafree-MC (Millipore, Billerica, MA, USA). The filtered solution was subjected to ultrafiltration using Amicon Ultra YM-10 (Millipore) and buffer-exchanged by 200 mM triethyl ammonium bicarbonate (TEAB; Sigma-Aldrich). The proteins were reduced by BKM120 adding 10 mM tris-(2-carboxyethyl)phosphine (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 55°C for 1 h. After the reaction, 20 mM iodoacetamide was added to the solution, and incubated for 30 min. The reactant was mixed with 1 mL of ice-cold acetone and incubated at −20°C for 3 h to precipitate proteins. The precipitated proteins were resuspended with 100 μL of 200 mM TEAB and mixed with 2 μl (1 μg μL-1) of sequencing grade ATM/ATR inhibitor modified trypsin (Promega, Madison, WI, USA) at 37°C overnight. The peptide concentration of the tryptic digests was measured using Protein Assay Bicinchoninate Kit (Nacalai tesque). The concentrations of the injected digests were 1.06 ± 0.12 μg μL-1 digest for free-living
M. loti and 4.96 ± 0.90 μg μL-1 digest for nodules, respectively. (mean ± SD, N = 3). LC-MS/MS analysis Proteome analyses were performed by a liquid chromatography (UltiMate3000 RSLCnano system (Thermo Fisher Scientific))/mass spectrometry (LTQ Velos mass spectrometer (Thermo Fisher Scientific)) system equipped with a long monolithic silica capillary column (200-cm long, 0.1-mm
ID) [24, 27]. 10 and 5 μL of tryptic digests were injected for free-living and symbiotic conditions, respectively, and separated by reversed-phase chromatography at a flow rate of 500 nL min-1. The gradient was provided Chlormezanone by changing the mixing ratio of the 2 eluents: A, 0.1% (v/v) formic acid and B, 80% (v/v) acetonitrile containing 0.1% (v/v) formic acid. The gradient was started with 5% B, increased to 50% B for 600 min, further increased to 95% B to wash the column, then returned to the initial condition, and held for re-equilibration. The separated analytes were detected on a mass spectrometer with a full scan range of 350–1,500 m/z. For data-dependent acquisition, the method was set to automatically analyze the top 5 most intense ions observed in the MS scan. An ESI voltage of 2.4 kV was applied directly to the LC buffer end of the chromatography column by using a MicroTee (Upchurch Scientific, Oak Harbor, WA, USA). The ion transfer tube temperature was set to 300°C. Triplicate analyses were done for each sample of 3 biological replicates, and blank runs were inserted between different samples.