Cell culture and medium
C. cellulovorans 743B (ATCC 35296) was grown anaerobically as described (Robert et al. [1984]) except for the carbon sources, which was 0.3% (w/v) cellobiose, 0.3% (w/v) avicel, or 0.3% (w/v) xylan.
Sample preparation of cellulosomal proteins for proteome analysis
Proteome samples were prepared from C. cellulovorans culture media. The culture (50 mL) was centrifuged (6,000 g, 25°C) and the supernatant was subjected to ultrafiltration using Amicon Ultra YM-10 (Millipore) to obtain the cellulosomal proteins (Adams et al. [2010]). The collected proteins were reduced with 10 mM tris(2-carboxyethyl)phosphine for 30 min and alkylated with 20 mM iodoacetamide for 60 min at room temperature. After acetone precipitation, the proteins were solubilized in 200 mM triethylammonium bicarbonate, trypsin-digested, and applied to a proteome analysis system.
Protein identification of cellulosomal proteins
Protein identification was performed by a liquid chromatography/mass spectrometry system. Proteolytic digests were separated by reversed-phase chromatography using a Prominence nano flow system (Shimadzu). A monolithic silica capillary column, prepared from a mixture of tetramethoxysilane and methyltrimethoxysilane (300 cm long, 0.1 mm ID) as described in (Motokawa et al. [2002]), was used at a flow rate of 500 nL/min. The gradient was provided by changing the mixing ratio of the 2 eluents; A, 0.1% (v/v) formic acid, and B, acetonitrile containing 0.1% (v/v) formic acid. The gradient was started with 5% B, increased to 45% B for 600 min, further increased to 95% B to wash the column, then returned to the initial condition, and held for re-equilibration. A packed tip column (NTCC-360, 150 mm × 100 μm I.D., Nikyo technos, Tokyo) was used as conventional packed column at a flow rate of 500 nL/min in gradient time 60 min. The separated analytes were detected on an LTQ Velos linear ion trap mass spectrometer (Thermo Scientific). For data-dependent acquisition, the method was set to automatically analyze the top 3 most intense ions observed in the MS scan. An ESI voltage of 2.4 kV was applied directly to the LC buffer distal to the chromatography column using a microtee. The ion transfer tube temperature on the LTQ Velos ion trap was set to 300°C. The mass spectrometry data were used for protein identification by Protein Discoverer software (Thermo Scientific) with the protein database built from genome analysis of C. cellulovorans (Tamaru et al. [2010a]). The data were then filtered at a q-value ≤ 0.01 corresponding to 1% FDR on a spectral level.