RNF26 Bridges ER Stress and Autophagy in the Clearance of MERS Envelope protein

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.