Shotgun Proteomics

Pellets of membrane-protected material were lysed with RIPA buffer containing quenchers (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 1x cOmplete Protease Inhibitor Cocktail (Roche), 1x PhosSTOP 956 (Roche), 10 mM sodium ascorbate, 1 mM Trolox and 1 mM sodium azide), sonicated and centrifuged at 10,000x g for 10 min. The supernatant was incubated with Streptavidin-agarose (Sigma-Aldrich) overnight, which was balanced with RIPA buffer containing quenchers. After 3x wash with RIPA buffer and 3x wash with 3 M Urea dissolved in 50 mM NH4HCO3, beads were incubated TCEP (5 mM, Sigma-Aldrich) at 55 °C for 30 min and shaken at 1000x rpm. Samples were alkylated with IAA (10 mM, Sigma-Aldrich) at room temperature for 20 min and shaken at 1000x rpm, further quenched by DTT (20 mM, Sigma-Aldrich) and washed 2x with 2 M Urea dissolved in 50 mM NH4HCO3. After overnight incubation with trypsin (1 μg/20 μl beads, Promega), supernatants were collected, plus 2x washes with 2 M Urea buffer. The samples were 966 acidified with trifluoroacetic acid (1%) and underwent vacuum centrifugation to decrease the volume. After being desalted on C18 stage tips (Thermo Scientific), peptides were reconstituted with 0.5% acetic acid for mass spectrometry analysis.