Samples were dissolved with chaotropic lysis buffer containing 8.4 M urea (USB Corporation, Cleveland, OH), 2.4 M thiourea (Sigma-Aldrich), 2.5 % SDS (Sigma-Aldrich), 5 mM TCEP (Sigma-Aldrich) and a protease inhibitor cocktail (Sigma-Aldrich), and incubated for 15 min on ice. Homogenization of the pellet was achieved by ultrasonication for 5 min on ultrasonic bath Branson 2510 (Marshall Scientific, New Hampshire, USA). The homogenate was centrifuged at 20000 × g for 10 min at 4 °C, and the supernatant containing the solubilized proteins was used for further analysis. Then, 20 μg of protein was precipitated by methanol/chloroform method and re-suspended in 20 μl of multichaotropic sample solution UTT buffer (7 M urea, 2M thiourea, 100mM TEAB (Sigma-Aldrich).
The re-suspended sample was reduced with 2 μL of 50 mM TCEP, pH 8.0, at 37 °C for 60 min, followed by addition of 1 μL of 200 mM cysteine-blocking reagent MMTS (SCIEX, Foster City, CA) for 10 min at room temperature. Sample was diluted to 140 μL to reduce the urea concentration with 25 mM TEAB. Finally, digestion was initiated by adding 1 μg of Pierce MS-grade trypsin (Thermo-Fisher Scientific Inc.) to each sample in a ratio 1:20 (w/w), and then incubated at 37°C overnight on a shaker. Sample digestion was evaporated to dryness in a vacuum concentrator.
The re-suspended sample was reduced with 2 μL of 50 mM TCEP, pH 8.0, at 37 °C for 60 min, followed by addition of 1 μL of 200 mM cysteine-blocking reagent MMTS (SCIEX, Foster City, CA) for 10 min at room temperature. Sample was diluted to 140 μL to reduce the urea concentration with 25 mM TEAB. Finally, digestion was initiated by adding 1 μg of Pierce MS-grade trypsin (Thermo-Fisher Scientific Inc.) to each sample in a ratio 1:20 (w/w), and then incubated at 37°C overnight on a shaker. Sample digestion was evaporated to dryness in a vacuum concentrator.