30k amicon filter

Cell pellets were lysed with lysis buffer (4% SDS and 2 mM TCEP in 0.1 M Tris pH 8.5). Protein concentration was measured by a BCA-reducing compatible kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein digestion was performed using a filter-aided sample preparation (FASP) procedure as described previously [80 (link),81 (link)]. After 200 μg of proteins was precipitated overnight at −20 °C using ice-cold acetone, protein digestion was performed via the two-step FASP procedure as described with some modifications [80 (link),81 (link)]. Protein pellets were dissolved in SDT buffer (4% SDS, 10 mM TCEP, and 50 mM CAA in 0.1 M Tris pH 8.0) and loaded onto a 30 K Amicon filter (Millipore, Jaffrey, NH, USA). The buffer exchanges were performed with UA solution (8 M urea in 0.1 M Tris pH 8.5) via centrifugation at 14,000× g for 15 min. Following the exchange of buffer with 50 mM TEAB, protein digestion was performed at 37 °C overnight using a trypsin/Lys-C mixture (Promega, Madison, WI, USA) at a 100:1 protein-to-protease ratio. The digested peptides were collected by centrifugation. After the filter units were washed with 40 mM ABC, the second digestion was performed at 37 °C for 2 h using trypsin (enzyme-to-substrate ratio (w/w) of 1:1000). The peptide concentration was measured by tryptophan assay [82 (link)].