2 cloroacetamide caa

The cell pellets were incubated with 6 ml of preheated SDC buffer containing 1% sodium deoxycholate (SDC, Sigma–Aldrich), 40 mM 2-cloroacetamide (CAA, Sigma–Aldrich), 10 mM tris(2-carboxyethyl)phosphine (TCEP; Thermo Fisher Scientific) and 100 mM Tris, pH 8.0. After incubation for 2 min at 95 °C, the samples were ultrasonicated for 2 min with 0.5 seconds pulse (50% intensity) and 0.2 s pause (Sonopuls, Bandelin). Incubation and ultrasonication was repeated for a second time. After a final incubation for 2 min at 95 °C, 1/6 of the sample was diluted 1:2 with MS grade water (VWR). Proteins were digested overnight at 37 °C with 50 μg trypsin (Promega). The solution of peptides was then acidified with trifluoroacetic acid (Merck) to a final concentration of 1%, followed by desalting via Sep-Pak C18 5cc vacuum cartridges (Waters). The cartridge was washed twice with 1 ml of 100% methanol, twice with 1 ml of 0.1% FA in 80% ACN and twice with 1 ml of 0.1% FA in water prior to sample loading. After loading the acidified sample, the cartridge was washed twice with 1 ml of 0.1% FA in water. Elution was done with 2 × 1 ml of 0.1% FA in 80% ACN.

For reduction and alkylation of the bound proteins, previously washed anti-FLAG M2 resins or the anti-HA magnetic resins were incubated with SDC buffer containing 1% sodium deoxycholate (SDC, Sigma-Aldrich), 40 mM 2-cloroacetamide (CAA, Sigma-Aldrich), 10 mM Tris (2-carboxyethyl) phosphine (TCEP, Thermo Fisher Scientific) and 100 mM Tris, pH 8.0 at 37 °C. After incubation for 20 min at 37 °C, the samples were diluted 1:2 with MS grade water (VWR). Proteins were digested overnight at 37 °C with 0.5 µg trypsin (Promega). The beads were centrifuged and the supernatant was collected. In addition, the beads were washed with 100 µL of buffer A (0.1% formic acid (Roth) in MS grade water (VWR)), followed by centrifugation and collection of the supernatant. The combined supernatants were acidified with trifluoroacetic acid (TFA, Merck) to a final concentration of 1%. Precipitated SDC was removed by centrifugation and the peptide mixture was desalted via SCX StageTips^{54 (link)}. Samples were vacuum dried and re-suspended in 10 µl of buffer A.