| 描述信息 |
Cells were harvested and lysed in 300 µL ice-cold lysis buffer using sonication. The lysates were clarified by dual centrifugation to remove debris. Protein concentration was determined via BCA assay using a microplate reader, with standard curve interpolation for quantification. Aliquots were stored at -80°C until use. For SDS-PAGE analysis, 10 µg protein/sample was resolved on 12% gels, stained, and imaged. For proteomic processing, 100 µg protein was denatured in 120 µL reduction buffer, alkylated with 50 mM iodoacetamide, and purified via 10 kDa ultrafiltration. Filters were washed twice with 300 mM TEAB before tryptic digestion (1 µg trypsin/sample, 37°C, 12 h). Peptides were eluted with 200 mM TEAB, lyophilized, and stored at -80°C.Peptide solutions were acidified to pH 7 with 1%formic acid. Desalting was performed using SOLA SPE 96-well plates preconditioned with 200 μL methanol and 200 μL H2O. Peptides were loaded twice, washed with 5% methanol, and eluted with 150 μL 100% methanol before lyophilization. LC-MS/MS analysis was conducted on a Q-Exactive mass spectrometer with a Nanospray Flex ion source. Peptides were separated on a C18 column using an EASY-nLC 1200 system with a 300 nL/min flow rate over 75 min. The gradient consisted of: 5-28% B, 28-42% B, 42-90% B , and 90% B. Full MS scans were acquired at 70,000 resolution. The top 10 most intense ions were selected for HCD fragmentation, with MS/MS spectra collected at 17,500 resolution. Dynamic exclusion was set to 30 s in positive ion mode |
