Protein Preparation, TMT-Labeling and LC-MS/MS Analysis

Cells subjected to different treatments (BP, HP) were collected for protein extraction. Briefly, cells were transferred into 2 mL low protein binding tubes and lysed with 600 μL extraction buffer [Sucrose: 2.4 g, NaCl 0.058 g, EDTA⋅2Na 0.146 g, DTT 0.02 g, 0.5 M Tris-HCl (pH6.8) 2.5mL, 0.5 M Tris-HCl (pH8.8) 2.5mL, and add ddH₂O to 10 mL] supplemented with PMSF to a final concentration of 1 mM. After lysis with sonication (80 W and 60 Hz for 3 min), samples were added with the same volume of Tris-phenol buffer (pH 7.8) and mixed for 30 min at 4°C. Then, the mixtures were centrifuged (8000 × g for 10 min at 4°C) to collect phenol supernatants. The supernatants were added with 5-fold volume of 0.1 M cold ammonium acetate-methanol buffer and precipitated at −20°C overnight. After precipitation completely, the samples were centrifuged (12000 × g for 10 min at 4°C) to collect precipitations. The precipitations were washed with cold methanol and then centrifuged (12000 × g for 10 min at 4°C) again to collect precipitations, repeated once. Then methanol was removed by washing twice with acetone. Then, the precipitations were collected with centrifugation at 12000 × g for 10 min at 4°C and dried at room temperature for 3 min, and then dissolved in lysis buffer (250 mM HEPES, 2% SDS, pH 7.0) for 3 h. Finally, the samples were centrifuged at 12000 × g for 10 min at room temperature to collect supernatants, repeated once. Protein concentration was determined by BCA assay (Thermo Scientific, United States).

Protein samples (50 μg) extracted from different groups of cell samples (BP, HP) were added with DTT to a final concentration of 5 mM and then incubated at 55°C for 30 min. Then iodoacetamide was added to a final concentration of 10 mM at room temperature in the dark for 15 min. Then the precooled acetone was used to precipitate the protein at −20°C overnight. After precipitation, the protein was collected by centrifugation (8000 × g for 10 min at 4°C). The enzymolysis diluent [protein:enzyme = 50:1 (m/m)] was added to redissolve the protein and then incubated for digestion with trypsin overnight at 37°C. Finally, samples were lyophilized after enzymolysis.

For TMT labeling, samples prepared above were resuspended in 50 μL 100 mM TEAB buffer (pH 8.0) in 1.5 ml tubes for labeling. Then 41 μL of TMT label reagent (Thermo Scientific, United States) was added to each sample for mixing at room temperature for 1 h. Finally, 8 μL of 5% hydroxylamine were added to terminate the reaction by incubation for 15 min.

Reversed-phase (RP) separation was performed on an 1100 HPLC System (Agilent). Mobile phases A (2% acetonitrile in HPLC water) and B (98% acetonitrile in HPLC water) were used for RP gradient. The solvent gradient was set as follows: 0∼8 min, 89% A; 8∼8.01 min, 98∼95% A; 8.01∼48 min, 95∼75% A; 48∼60 min, 75∼60% A; 60∼60.01 min, 60∼10% A; 60.01∼70 min, 10% A; 70∼70.01 min, 10∼98% A; and70.01∼75 min, 98% A. Tryptic peptides were separated at a fluent flow rate of 300 μL/min and monitored at 210 and 280 nm. Samples were collected for 8–60 min, and the eluent was collected in a centrifugal tube. Samples were recycled in this order until the end of the gradient. Subsequently, the lyophilized peptides were analyzed by tandem mass spectrometry (MS/MS) using Q-Exactive mass spectrometer (Thermo, United States) equipped with a Nanospray Flex source (Thermo, United States). Full MS scans were acquired in the mass range of 350–1500 m/z with a mass resolution of 60,000 and the AGC target value was set at 3e⁶. The 10 most intense peaks in MS were fragmented with higher-energy collisional dissociation (HCD) with an NCE of 32. MS/MS spectra were obtained with a resolution of 15,000 with an AGC target of 2e⁵ and a max injection time of 40 ms. The Q-E dynamic exclusion was set for 30.0 s and run under positive mode.