Cell Lysis and Protein Digestion Protocol

When harvesting cells, medium was removed from each well, and cells were carefully washed twice with cold PBS. Lysis buffer (0.15 mL, 50 mM HEPES, pH 8.5, fresh 8 M urea, 0.5% sodium deoxycholate (NaDoc), and 1 mM DTT) was subsequently added and liquid was transferred into a tube with glass beads for complete lysis in a Bullet Blender. The protein concentration was measured by BCA (Thermo Scientific, Ref No 23250) with BSA as a standard. Protein digestion was performed as previously reported^{22 (link)}. Briefly, 100 μg proteins were proteolyzed with Lys-C (peptide: enzyme = 1:100 w/w) for 2 hrs and further digested by trypsin (1:50 w/w) overnight at room temperature in 2 M urea. Following digestion, the digested peptides were reduced and alkylated before being desalted by C18 spin-columns (Harvard Apparatus, #74–7206) according to the manufacturer’s instruction.