Proteomic raw data
Description
To investigate the proteomic changes induced by dACSL4, SH-SY5Y cells were treated with 1 μM dACSL4 for 12 h. The cell lysates (50 μg) were then boiled (95°C for 5 min) and separated by a gradient SDS-PAGE (4%-20%) gel and electrophoresed at 150 V for 10 min. The gel was subsequently stained with Coomassie Brilliant Blue, and each lane was excised separately. Each lane of protein gel was cut into granules before being put into a clean centrifuge tube respectively, 500 μL 50% acetonitrile in 50 mM NH4HCO3 was added, and the solution was placed at 37°C until the gel color faded. The blue solution was discarded, and enough acetonitrile was added to allow for gel dehydration. The dry gel granules were reduced with 5 mM dithiothreitol and alkylated with 11 mM iodoacetamide, followed by adding enough acetonitrile to allow for gel dehydration again. The dry gel granules were added to a moderate trypsin solution (10 ng μL-1 trypsin in 50 mM NH4HCO3) and incubated at 37°C for 16 h. Peptides in solution and gel were extracted three times with 0.1% trifluoroacetic acid (TFA) in a 50% acetonitrile aqueous solution for 1 h. Extracts were then combined and dried by vacuum centrifugation. Dry tryptic peptides were dissolved in 20 μL 0.1% TFA in water and analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). For HPLC-MS/MS analysis, the peptides were separated and identified by a 2 h gradient elution at a flow rate 0.30 μL min-1 with a Thermo-Dionex Ultimate 3000 HPLC and a Thermo Scientific Orbitrap Exploris 480 mass spectrometer combining system. The analytical column was a homemade fused silica capillary column (75 µm ID, 350 mm length) packed with C18 resin (1.9 µm, Dr. Maisch GmbH). Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 80% acetonitrile and 0.1% formic acid. The Orbitrap Exploris 480 mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 4.5.445.18 software and there was a single full-scan mass spectrum in the Orbitrap (350-1600 m/z, 60,000 resolution) followed by followed by 2 seconds data-dependent MS/MS scans in the Ion Routing Multipole with a normalized collision energy of 30 (HCD). Adsorbed proteomics were identified and quantified using label-free quantification method by running MaxQuant_2.6.7.0 against the Uniprot Swiss-Prot database of human (downloaded on 18th, September, 2025). The search criteria were as follows: trypsin was chosen as the specific enzyme; up to two missed cleavages were allowed; carbamidomethylation (C) was set as the fixed modification; precursor ion mass tolerances were set at 20 ppm for all MS acquired in an Orbitrap mass analyzer; and the fragment ion mass tolerance was set at 0.02 Da for all MS2 spectra. Confidence levels were set to 1% FDR (high confidence).
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Institutions
- Institute of ChemistryBeijing, Beijing