DOI: 10.57760/sciencedb.08403
title: Proteome data of RAW264.1 cells (label-free)
title: Proteome data of RAW264.1 cells (label-free)

description: RAW 264.7 cells were seeded at 3×104 cells/mL in T75 flask and pre-incubated with QEPV (0, 0.3 mg/mL) in DMEM culture medium for 48 hours. Cells were then exposed to H2O2 at 0 or 0.1 mM for 15 minutes, washed twice with PBS, scraped off using cell scrapers, collected at 1000 g for 3 minutes, and resuspended in 1 mL PBS. Cells were stored in liquid nitrogen for proteomics analysis.Protein in the cells was extracted using BeyoLytic™ Mammalian Active Protein Extraction Reagent (Beyotime, China) on ice. Then protein was reduced with 10 mM dithiothreitol (DTT) at 37 °C for 60 min. After cooling to room temperature, alkylation was performed with 50 mM iodoacetamide (IAA) at room temperature in the dark for 60 min. Extracts were diluted 5-fold in 25 mM ammonium bicarbonate (AmBic), to a concentration of 1.6 M urea for tryptic digestion at 37 °C for 20 hours. Peptides were freeze-dried, redissolved in 0.1% fomic acid, and desalinized by C18 Cartridge (WAT023590, Waters).The peptides from tryptic digestion were separated by Easy nLC/ Ultimate 3000 instrument (Thermo Scientific, USA). A binary mobile phase consisted of 0.1% formic acid in water (A) and 80% acetonitrile in 0.08% formic acid (B). A flow rate of 600 nL per minute was used to load the sample onto a pre-column of C18 3 mm 100 μm×20 mm and a C18 1.9 mm 150 μm×120 mm column. The gradient was 6% solvent B from 0-10 min, 6–10% B from 10-15 min, 10–30% B from 15-70 min, 30-40% B from 70-80 min, and 40-95% B from 80.1-85 min.The LC effluent was introduced into a Q Exactive HF-X (Thermo Fisher Scientific, USA) using the manufacturer's nano-electrospray ion source. The analysis time was 88 minutes, utilizing the positive ion detection method with a scanning range of parent ions from 300 to 1550 m/z. The primary mass spectrometry resolution is set at 120000, with a maximum IT of 20 ms. The dynamic exclusion time was set at 15.0 s. For obtaining the mass charge ratio of polypeptides and polypeptide fragments, the following methods were employed: MS2 Activation Type to be HCD, isolation window 1.6 m/z, secondary mass spectroscopic resolution 15000 at 110 m/z. Microscans to be 1 and secondary Maximum IT to be 30 ms. Normalized Collision Energy was maintained at 27 eV.The identification of peptides was performed with Proteome Discoverer (v2.5, Thermo Fisher Scientific) against the Uniprot mouse reference database (UP000000589_10090 _20231127.fasta). The fold change (FC) of proteins &lt; 0.05 or &gt; 2 were selected, and cell pathway analysis was performed with KEGG. Results were considered significant at p &lt; 0.05 in all analyses performed.
description: RAW 264.7 cells were seeded at 3×104 cells/mL in T75 flask and pre-incubated with QEPV (0, 0.3 mg/mL) in DMEM culture medium for 48 hours. Cells were then exposed to H2O2 at 0 or 0.1 mM for 15 minutes, washed twice with PBS, scraped off using cell scrapers, collected at 1000 g for 3 minutes, and resuspended in 1 mL PBS. Cells were stored in liquid nitrogen for proteomics analysis.Protein in the cells was extracted using BeyoLytic™ Mammalian Active Protein Extraction Reagent (Beyotime, China) on ice. Then protein was reduced with 10 mM dithiothreitol (DTT) at 37 °C for 60 min. After cooling to room temperature, alkylation was performed with 50 mM iodoacetamide (IAA) at room temperature in the dark for 60 min. Extracts were diluted 5-fold in 25 mM ammonium bicarbonate (AmBic), to a concentration of 1.6 M urea for tryptic digestion at 37 °C for 20 hours. Peptides were freeze-dried, redissolved in 0.1% fomic acid, and desalinized by C18 Cartridge (WAT023590, Waters).The peptides from tryptic digestion were separated by Easy nLC/ Ultimate 3000 instrument (Thermo Scientific, USA). A binary mobile phase consisted of 0.1% formic acid in water (A) and 80% acetonitrile in 0.08% formic acid (B). A flow rate of 600 nL per minute was used to load the sample onto a pre-column of C18 3 mm 100 μm×20 mm and a C18 1.9 mm 150 μm×120 mm column. The gradient was 6% solvent B from 0-10 min, 6–10% B from 10-15 min, 10–30% B from 15-70 min, 30-40% B from 70-80 min, and 40-95% B from 80.1-85 min.The LC effluent was introduced into a Q Exactive HF-X (Thermo Fisher Scientific, USA) using the manufacturer's nano-electrospray ion source. The analysis time was 88 minutes, utilizing the positive ion detection method with a scanning range of parent ions from 300 to 1550 m/z. The primary mass spectrometry resolution is set at 120000, with a maximum IT of 20 ms. The dynamic exclusion time was set at 15.0 s. For obtaining the mass charge ratio of polypeptides and polypeptide fragments, the following methods were employed: MS2 Activation Type to be HCD, isolation window 1.6 m/z, secondary mass spectroscopic resolution 15000 at 110 m/z. Microscans to be 1 and secondary Maximum IT to be 30 ms. Normalized Collision Energy was maintained at 27 eV.The identification of peptides was performed with Proteome Discoverer (v2.5, Thermo Fisher Scientific) against the Uniprot mouse reference database (UP000000589_10090 _20231127.fasta). The fold change (FC) of proteins &lt; 0.05 or &gt; 2 were selected, and cell pathway analysis was performed with KEGG. Results were considered significant at p &lt; 0.05 in all analyses performed.