Tc18 sep pak vac cartridge

After the click-chemistry reaction, the proteins were reduced with 5 mM dithiothreitol at 56 °C for 30 min, alkylated with 14 mM iodoacetamide at room temperature in the dark for 30 min, and purified with the methanol-chloroform precipitation method. Proteins were digested with sequencing-grade modified trypsin (Promega) in the buffer containing 1.6 M urea, 50 mM HEPES pH 8.8, and 5% acetonitrile (ACN), which lasted for 16 h with shaking at 37 °C. The digestion was quenched with 0.4% trifluoroacetic acid (Millipore), and the pH was checked to be lower than 2. The peptides were desalted using a tC18 Sep-Pak Vac Cartridge (Waters) and dried using a vacuum concentrator.

The cells were lysed and the OPP-labeled nascent proteins were enriched through the copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction. For the background sample, the catalytic reagents (CuSO₄ and THPTA) were not added to the solution. The reaction lasted for 2 h in the dark and was quenched by adding 10 mM dithiothreitol (DTT; Sigma-Aldrich). The enriched proteins were further reduced at 56 °C for 25 min and alkylated with 14 mM iodoacetamide at room temperature in the dark for 30 min. The beads from all samples were stringently washed to remove the nonspecific binding proteins. The enriched newly synthesized proteins were digested on beads with trypsin (Promega) at 37 °C overnight. The digestion was quenched with trifluoroacetic acid (Millipore), and the pH was adjusted to ~2 before desalting. The supernatant containing eluted peptides was collected, and the peptides were purified using a tC18 Sep-Pak Vac Cartridge (Waters). The purified peptides were dried in a vacuum concentrator. The detailed information is included in the Supporting Information.