Pierce ms grade chymotrypsin protease

The samples were split for tryptic and chymotryptic digestion and processed using a modified protocol of FASP (33 (link)). Briefly, triethylammonium bicarbonate (TEAB) was added to a final concentration of 50 mM TEAB before reduction using 100 mM dithiothreitol at 56°C for 30 min. The reduced samples were loaded onto 10-kDa molecular weight cutoff of Pall Nanosep centrifugal filters (Sigma-Aldrich), washed with 8 M urea and 1% sodium deoxycholate (SDC), and alkylated with 10 mM methyl methane thiosulfonate. Two-step digestion was performed on filters using trypsin and chymotrypsin as digestive enzymes in 50 mM TEAB and 0.5% SDC buffer. The first step was performed overnight, and the second step, with an additional portion of proteases, was performed for 4 hours the next day. Tryptic digestion was performed at 37°C using Pierce MS Grade Trypsin Protease (Thermo Fisher Scientific). Chymotryptic digestion was performed at room temperature using Pierce MS Grade Chymotrypsin Protease (Thermo Fisher Scientific). The peptides were collected by centrifugation, and SDC was precipitated by acidifying the sample with trifluoroacetic acid (final concentration, 1%). The digested sample was desalted using Pierce Peptide Desalting Spin Columns (Thermo Fisher Scientific) according to the manufacturer’s protocol.