Azide magnetic beads

The SK-N-BE(2 (link)) cell lysates in 0.2% SDS with 1X protease inhibitor cocktail were pretreated with 120 μM bufalin (37°C, 2 h) followed by 120 μM CS-P1 (37°C, 4 h) or DMSO (37°C, 4 h). After incubation on ice for 30 min and sonication at 4°C for 2 min at 30%, the lysates (400 μg total protein) were added to freshly prepared click reaction cocktail [10 μl azide magnetic beads (Click Chemistry Tools), 1 mM CuSO₄, 2 mM TCEP and 80 μM TBTA] for 1.5 h at room temperature with rotation. After reductive alkylation [100 mM ammonium bicarbonate (NH₄hco₃; Sangon Biotech Co., Ltd.) with 20 mM TCEP and 40 mM iodoacetamide (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature in the dark], the beads were washed twice with 8 M urea, followed by three washes with PBS. Next, the beads were resuspended with 200 μl 100 mM NH₄hco₃ containing 4 μg trypsin (Promega Corporation) and incubated at 37°C overnight. Finally, the supernatant was collected and desalted using a ZipTip with 0.6 μl C18 column (5 μg; cat. no. ZTC18S960; Millipore). After drying in a speed-vac, samples were stored at -80°C prior to MS analysis. Each treated sample was prepared in three biological replicates.