Eksigent nanolc ultra 2d

An E. coli strain was transformed with pSHK5(TS)-NgAgo-Flag, pSHK5(TS)-NgAgo fragments (A, B or D)-Flag, or pSHK5(TS). The bacteria were collected in 2 ml RIPA buffer (Beyotime), and then lysed by two passages through a French pressure cell (Thermo, USA) at 20 000 lb/in.². After centrifugation at 12 000g for 10 min, the supernatants were collected and further purified with FLAG M Purification Kit (Sigma-Aldrich, CELLMM2). Finally, the interacting proteins were recovered with PBS buffer containing 300 μg/ml of 3× FLAG peptides (Sigma-Aldrich, F4799). The eluted proteins were subjected for analysis by LC-MS/MS with Eksigent nanoLC-Ultra 2D (AB SCIEX) and TripleTOF 5600 (AB SCIEX). The data analysis was performed with the Mascot 2.3 (Matrix Science) and was based on the SwissProt 57.15 database (Taxonomy: E. coli) with the following parameters: Type of search: MS/MS Ion Search; Mass values: Monoisotopic; Fixed modifications: Carbamidomethyl (C); Variable modifications: Acetyl (Protein N-term), Deamidated (NQ), Dioxidation (W) and Oxidation (M); Peptide mass tolerance: ±30 ppm; Fragment mass tolerance: ±0.15 Da; and Max missed cleavages: 2. To ensure the reliability of identified proteins, proteins that more than two unique peptides were successfully matched were recognized as the identified proteins.