Easy nlc 1000 liquid chromatography

Total protein was extracted and determined using the Lowry method. For peptide preparation, 10 µg of protein was separated using SDS-PAGE before dehydration with acetonitrile. Proteins were treated with 10 mM DTT (Merck KGaA, Darmstadt, Germany) at 56 °C for 1 h to reduce the sulfhydryl group and incubated with 100 mM iodoacetamide (Sigma-Aldrich, St. Louis, MO, USA) in the dark for 45 min for alkylation. The gel was then dehydrated by incubating in acetonitrile for 5 min before digestion with trypsin (Promega, Mannheim, Germany) at 37 °C for 16–18 h. The tryptic peptides were then subjected to dimethyl labeling before loading to the EASY-Spray™ C18, 75 cm × 75 μm column (Thermo Fisher Scientific, Waltham, MA, USA) and separated with the gradient using mobile phase A (0.1% formic acid in LC-MS-grade water) and mobile phase B (0.1% formic acid in LC-MS-grade acetonitrile) for 90 min with a flow rate of 300 nL/min. Cellular proteins were analyzed using LC-MS/MS operated on the EASY-nLC 1000 liquid chromatography (Thermo Fisher Scientific) and the Q exactive™ plus hybrid quadrupole-orbitrap™ mass spectrometer (Thermo Fisher Scientific). Proteins were then identified based on their spectra using Proteome Discoverer 2.1 software (Thermo Fisher Scientific) and the human UniProt database (https://www.uniprot.org/, accessed on 20 October 2022).