C18 solid phase extraction spin columns

Samples rested at RT for 20 minutes before heating to 99°C for 5 min. FASP was performed using a 30 kDa molecular weight cutoff filter (VIVACON 500; Sartorius Stedim Biotech GmbH, 37070 Goettingen, Germany) essentially according to the procedure described by Wisniewski et al.^{52 (link)}. Fifty µl of each cleared protein extract was mixed with 200 μl of freshly prepared 8 M urea in 100 mM Tris-HCl (pH 8.5) (UA-solution) in the filter unit and centrifuged at 14,000 g for 15 min at 20°C to remove SDS. Any residual SDS was washed out by a second washing step with 200 μl of UA. The proteins were alkylated with 100 μl of 50 mM iodoacetamide in the dark for 30 min at RT. Afterward, three washing steps with 100 μl of UA solution were performed, followed by three washing steps with 100 µl of 50 mM TEAB buffer (Sigma-Aldrich). Proteins were digested with 1.25 µg trypsin overnight at 37 °C. Peptides were recovered using 40 μl of 50 mM TEAB buffer followed by 50 μl of 0.5 M NaCl (Sigma-Aldrich). Peptides were desalted using C18 solid phase extraction spin columns (The Nest Group, Southborough, MA), organic solvent removed in a vacuum concentrator at 45°C and reconstituted in 5% formic acid and stored at -80°C until LC-MS/MS analysis (see Supplementary Methods, also for analyses of mass spectrometry data).