Typically, 0.65 g of lyophilized cell walls was used for one experiment. Proteins were extracted by successive salt solutions in this order: two extractions each time with 6 mL CaCl2 solution (5 mM acetate buffer, pH 4.6, 0.2 M CaCl2 and 10 μL protease inhibitor cocktail), followed by two extractions with 6 mL LiCl solution (5 mM acetate buffer, pH 4.6, 2 M LiCl and 10 μL protease inhibitor cocktail). Cell walls were resuspended by vortexing for 5–10 min at room temperature, and then centrifuged for 15 min at 4000 × g and 4°C. Supernatants were desalted using Econo-Pac® 10 DG columns (Bio-Rad) equilibrated with 0.2 formic acid ammonium salt. The extract were lyophilized and resuspended in sample buffer for separation of proteins by 1D-GE, as previously described [12 (link)].
The next extraction was carried out by SDS and DTT. The cell wall preparation was treated with 12 mL solution containing 62.5 mM Tris, 4% SDS, 50 mM DTT, pH 6.8 (HCl). The mixture was boiled for 5 min and centrifuged for 15 min at 40000 × g and 4°C. The supernatant was dialyzed against 1 L H2O in Spectra/Por® membrane 10 kDa MWCO bags (Spectrum Medical Industries) at room temperature, then concentrated by successive centrifugation using the Centriprep® centrifugal filter devices (YM-10 kDa membrane) (Millipore) at 4000 × g followed by speed vacuum centrifugation.
The protein content of each extract was measured using the Bradford method [27 (link)] with the Coomassie™ protein assay reagent kit (Pierce) using bovine serum albumin (BSA) as standard.
Gels were stained with Coomassie™ Brilliant Blue-based method [28 (link)]. Colored bands were digested with trypsin and MALDI-TOF MS or LC-MS/MS analyses were performed as previously reported [12 (link),13 (link)].
The sequences of the identified proteins were subsequently analyzed with several bioinformatic programs to predict their sub-cellular localization [29 -31 ]. In some cases, predictions were not the same with the three programs. Results are then indicated as "not clear". Data are described in Tables 1–5 (additional data).
The next extraction was carried out by SDS and DTT. The cell wall preparation was treated with 12 mL solution containing 62.5 mM Tris, 4% SDS, 50 mM DTT, pH 6.8 (HCl). The mixture was boiled for 5 min and centrifuged for 15 min at 40000 × g and 4°C. The supernatant was dialyzed against 1 L H2O in Spectra/Por® membrane 10 kDa MWCO bags (Spectrum Medical Industries) at room temperature, then concentrated by successive centrifugation using the Centriprep® centrifugal filter devices (YM-10 kDa membrane) (Millipore) at 4000 × g followed by speed vacuum centrifugation.
The protein content of each extract was measured using the Bradford method [27 (link)] with the Coomassie™ protein assay reagent kit (Pierce) using bovine serum albumin (BSA) as standard.
Gels were stained with Coomassie™ Brilliant Blue-based method [28 (link)]. Colored bands were digested with trypsin and MALDI-TOF MS or LC-MS/MS analyses were performed as previously reported [12 (link),13 (link)].
The sequences of the identified proteins were subsequently analyzed with several bioinformatic programs to predict their sub-cellular localization [29 -31 ]. In some cases, predictions were not the same with the three programs. Results are then indicated as "not clear". Data are described in Tables 1–5 (additional data).