| F0000000099 | |
| ID | F0000000099 |
| Title | SCX fraction |
| Subcellular | Whole |
| Protein | SCX |
| Peptide | Reverse phase |
| Note | Cells cultured in each condition were harvested by centrifugation at 7,000 × g for 15 min, then washed three times with PBS. The bacterial cells were resuspended in lysis buffer and disruption by ultrasonication for 15 min. The resultant suspension was centrifuged at 40,000 × g for 30 min and the protein concentrations in the collected supernatants were measured using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, USA). The cell protein extracts were reduced in 1 mM dithiothreitol (25°C for 20 min) and then alkylated in 5.5 mM iodoacetamide (25°C for 15 min, in the dark). Trypsin (Promega) was added to a final ration of 1:50 (V:V) and proteins were digested in solution overnight at 37°C. The acquired peptides were dried under vacuum, and stored at −80°C for further use. All experiments were performed on a LTQ Q-Exactive HF mass spectrometer (Thermo Scientific, USA) coupled online with a nano-HPLC (Ultimate 3000, Thermo Scientific). The nano-HPLC system was used for peptide separation equipped with C18 reversed-phase microcapillary trapping at a flow rate of 600 nL/min. The mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The peptides were eluted using a gradient (6–95% mobile phase B) during a 195 min LC run and then sprayed directly into the MS instrument. |