Ketamine-induced astrocyte proteome
Description
This dataset contains abundance of proteins found in astrocytes isolated from the cortex of adult male mice 24 hours after treatment with a subanesthetic dose of ketamine (10mg/kg) or equivalent volume of saline, administered via intraperitoneal injeciton. Mice were 4 months old male C57BL/6J. All animal work was approved by the Institutional Animal Care and Use Committee (IACUC) of Thomas Jefferson University. All animal experiments were conducted following ARRIVE guidelines. Mice were deeply anesthetized with 5% inhaled isoflurane, and transcardially perfused with icecold PBS. Astrocytes were isolated from the dissected cortices using the magentic-activated cell sorting (MACS) protocol from Miltenyi. Samples for LC-MS/MS were precipitated with ice cold acetone and digested with Trypsin/LysC in denatured conditions. The native digestion procedure is outlined in V.E.Hedrick, M.N. LaLand, E.S. Nakayasu and N.P.Lake. Digestion, Purification, and Enrichment of Protein Samples for Mass Spectrometry (2015) Curr. Protoc. Chem. Biol 7(3):201-222. Data acquisition was performed using the ThermoFisher Scientific Vanquish LC system coupled to the Orbitrap 480 Exploris (ThermoFisher Scientific, Waltham, MA). Following digestion, ~2ug of digested peptides were loaded onto a ThermoFisher PepMap Acclaim, 1 x 150 mm C18 analytical column heated to 50°C. Peptides were separated using a 125-minute gradient elution method at a flow rate of 80 μl/min. Mobile phase A consisted of 0.1% formic acid (FA) in water while mobile phase B consisted of 0.1 % FA in acetonitrile (ACN). The linear gradient started at 2% B and reached 32% B in 103 minutes. An additional linear gradient from 32% to 90% B was performed from 103 to 107 minutes to promote elution of hydrophobic peptides. The column was washed in 90% B from 107 to 112 minutes. The system was re-equilibrated to 2% B from 112 to 125 minutes.The samples were injected into the heated electrospray ion source (HESI). MS spectra were collected at a resolution of 120,000 and a scan range of 350-1800 m/z. Data dependent analysis (DDA) was performed using a normalized collision energy of 28% at a resolution of 15,000. Table 3 provides a description of the instruments and of the LCMS test conditions. Peptide identification was performed using Proteome Discoverer 3.1.1 for protein identification. Database search parameters included 2 miscleavages, 10 parts per million (ppm) mass tolerance, and fixed carbamidomethylation modification of cysteines (C). Oxidation, methylation and deamidation were included as variable modifications to prevent misidentification peptides. The false discovery rate (FDR) was set to 0.01 and the mouse proteome Uniprotkb 000000589 was searched using comet and Sequest HT as the search algorithms. In addition, a decoy database was searched to reduce false positives.
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Institutions
- Thomas Jefferson UniversityPennsylvania, Philadelphia