MassIVE MSV000086866

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Thymic epithelial cells from WT and AB0/0 mouse model

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Description

Murine TEC for proteomic analyses were obtained from 3 pools of WT or AB0/0 mice, 3 mice per pool (5-6 week-old). TEC were isolated, enriched and processed through CD45+ cell-depletion as described above. CD45- cell-samples were pelleted and stored at -80 C until use. Murine CD45- TEC samples were lysed through three quick freeze/thaw cycles and the protein extraction was performed by adding RapiGestTM SF at 0.2% (w/w) according to the manufacture s protocol (Waters Corporation, Milford, MA, USA). Enzymatic digestion was conducted by adding trypsin in a ratio 1:50 (w/w) o/n and then a further aliquot in a ratio 1:100 (w/w) was added for 4 h. Each peptide mixture was desalted by Pep-Clean C-18 spin columns (Pierce Biotechnology Inc., Rockford, IL, USA), concentrated at 60 C and finally reconstituted in 0.1% formic acid. After enzymatic digestion with trypsin, samples were analyzed using nano-chromatography coupled to orbitrap mass spectrometer (LC MS/MS). Globally, 3 biological samples per condition were analyzed in technical replicates (n=2). In particular, the chromatographic separation of peptides was performed using the Eksigent nanoLC-Ultra 2D System (Eksigent, part of SCIEX Dublin, CA, USA) combined with cHiPLC- nanoflex system (Eksigent) in trap-elute mode. Peptides were eluted in 100 min with an acetonitrile gradient (mobile phase A: 0.1% formic acid in water; mobile phase A: 0.1% formic acid in acetonitrile), ionized by a nanospray ionization source (EASY-SprayTM 90 Source, Thermo Fisher Scientific, San Jose, CA, USA) and analyzed using QExactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Full mass spectra were recorded in positive ion mode in a range of 400-1600 m/z, with a resolution of 70000 FWHM (@ m/z 200) and 1 microscan per second. Each full scan was followed by 7 MS/MS events (resolution of 17,500 FWHM) generated in a data dependent manner on the top seven most abundant isotope patterns with charge > 2 (isolation window of 2 m/z from the survey scan, normalized collision energy of 30 and dynamic exclusion of 30 sec). [doi:10.25345/C59Z1B]

Keywords: Thymus, mouse, MHCII, Epithelial cells, Bare Lymphocyte Syndrome

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Principal Investigators:
(in alphabetical order) 		Dario Di Silvestre, Institute of Biomedical Technologies, Italy
Francesca Brambilla, Institute of Biomedical Technologies, Italy
Pierluigi Mauri, Institute of Biomedical Technologies, Italy
Submitting User: Dds100
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