2026-10-14T11:00:13Z https://www.scidb.cn/oai

10.57760/sciencedb.38185 2026-06-05T15:01:59Z

2026-06-05 RNF26 Bridges ER Stress and Autophagy in the Clearance of MERS Envelope protein doi:10.57760/sciencedb.38185 en To elucidate the molecular mechanisms underlying MERS-E–induced autophagy, we performed quantitative proteomic analysis of cells overexpressing MERS-E compared with vector controls. The data suggest that MERS-E expression enhances the cellular capacity for protein turnover and clearance, in line with the observed instability of the MERS-E protein itself.H292 cells were washed three times with cold PBS buffer and then lysed in SDT lysis buffer (4% w/v SDS, 100mM Tris-HCl, 100mM DTT, pH 7.6). The cell lysates were incubated for 10 min at 95 ℃ and centrifuged at 15,000 ×g for 15 min. The supernatants were collected, and protein concentration was determined by tryptophan fluorescence emission assay as described previously(1). 100 μg of proteins from each sample were alkylated (350mM iodoacetamide, 45 min, 25 °C in the dark). Solvent precipitation SP3 (SP4) captures aggregated protein for proteomics sample clean-up(2). 1000 µg inert glass beads (water suspended) were added in bead/protein ration of 10:1 (w/w), then add 4 volumes of anhydrous Acetonitrile and centrifuge to remove 90-95% of the supernatant, wash with 80% ethanol for 3 times and dried in vacuum. Subsequently, trypsin was added to a 1:100 enzyme: protein ratio and digested overnight at 37 °C. The next day, 10% trifluoroacetic acid was added to stop the enzyme digestion, and the pH of the solution was adjusted to about 3.0. About 20 μg of peptides from each sample were desalted for LC-MS/MS analysis.LC-MS/MS analysis The DIA analysis was performed on an hybrid TIMS quadrupole TOF mass spectrometer (Bruker timsTOF Pro) platform connected to an online nanoflow U3000 HPLC system (Thermo Fisher Scientific, USA). For each sample, 100 ng of peptides were resolved using 0.1% formic acid and separated using a home-made reversed-phase C18 column (20 cm × 75 µm, ReproSil-Pur C18-AQ, 1.9 µm resin, Dr. Maisch GmbH, Germany) using a 60 min gradient at 300 nL/min. Mobile phase A consisted of 0.1% (v/v) formic acid in H2O and Mobile phase B consisted of 0.1% (v/v) formic acid in 80% acetonitrile. Liquid chromatography was coupled online to a hybrid TIMS quadrupole TOF mass spectrometer (Bruker timsTOF Pro) via a CaptiveSpray nano-electrospray ion source. The source parameters were: 1500 V of Capilary voltage, 3.0 L/min of dry gas, and 180 °C of dry temperature. To perform data-independent acquisition, samples were acquired with a diaPASEF method106 consisting on 14 cycles including a total of 28 mass width windows (25 Da width, from 452 to 1,152 Da) with 4 mobility windows each, making a total of 56 windows covering the ion mobility range (1/K0) from 0.76 to 1.29 V s/cm2. Database searching and data analysis Raw data were processed using Spectronaut software (3) against the Uniprot human proteome database, with precursor FDR at 1%. Carbamidomethyl were selected as fixed modifications and N-terminal methionine excision enabled. Peptide length range was 7-30 and one missing cleavage site was allowed. LC-MS/MS; MERS-E; RNF26 李紫月 王扬 郄静波 李双渠 高召兵 朱轩 夏冰清 PUBLIC https://creativecommons.org/licenses/by/4.0/ dataset http://www.doi.org/10.1016/j.virs.2026.06.005 Science Data Bank