SDS-PAGE and Mass Spectrometry

Wild-type SM9913 strain was grown in SWLB medium at 15 or 37°C for 24 h, respectively. The cultures of 5 mL were centrifuged at 13,000 rpm for 2 min and the supernatants were removed. Cell pellets were re-suspended in 1 mL lysis buffer [50 mM Tris (pH 8.0), 100 mM NaCl, and protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, United States)]. Then samples were sonicated twice at level 2 for 5 min using a Sonic Dismembrator (Ningbo Scientz Biotechnology, Co., Ltd., Ningbo, China). The supernatant was collected after centrifugation at 13,000 rpm for 5 min and the protein concentration was measured using a Bi Yuntian BCA assay kit (Beyotime Biotechnology, Co., Ltd., Haimen, China). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed by loading 25 μg of each sample. Gels were stained with Coomassie Brilliant Blue R-250 and 10 protein bands were cut and subjected to in-gel tryptic digestion. In-gel digestion and mass spectrometry (MS) analysis of the gel slices were performed at the Research Center for Life Sciences University of Science and Technology (Hefei, Anhui, China) as previously described (Shevchenko et al., 2006).