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Thymic epithelial cells from WT and AB0/0 mouse model

ABSTRACT: 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).

INSTRUMENT(S): QExactive

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Francesca Brambilla Pierluigi Mauri Dario Di Silvestre

PROVIDER: MSV000086866 | MassIVE | Mon Feb 15 05:48:00 GMT 2021

REPOSITORIES: MassIVE

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Project description:The Hippo pathway, which is conserved from Drosophila to mammals, has been recognized as a tumor suppressor signaling pathway governing cell proliferation and apoptosis, two key events involved in organ size control and tumorigenesis. Although several upstream regulators, the conserved kinase cascade and key downstream effectors including nuclear transcriptional factors have been defined, the global organization of this signaling pathway is not been fully understood. Thus, we conducted a proteomic analysis of human Hippo pathway, which revealed the involvement of an extensive protein-protein interaction network in this pathway. Our data suggest that 550 interactions within 343 unique protein components constitute the central protein-protein interaction landscape of human Hippo pathway. Our study provides a glimpse into the global organization of Hippo pathway, reveals previously unknown interactions within this pathway, and uncovers new potential components involved in the regulation of this pathway. Understanding these interactions will help us further dissect the Hippo signaling-pathway and extend our knowledge of organ size control. Mass spectrometry data anaylsis: Excised gel bands were cut into approximately 1 mm3 pieces. Gel pieces were then subjected to in-gel trypsin digestion and dried. Samples were reconstituted in 5 ul of HPLC solvent A (2.5% acetonitrile, 0.1% formic acid). A nano-scale reverse-phase HPLC capillary column was created by packing 5 um C18 spherical silica beads into a fused silica capillary (100 um inner diameter x 20 cm length) with a flame-drawn tip. After equilibrating the column each sample was loaded via a Famos autosampler (LC Packings, San Francisco CA) onto the column. A gradient was formed and peptides were eluted with increasing concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid). As peptides eluted they were subjected to electrospray ionization and then entered into an LTQ Velos ion-trap mass spectrometer (ThermoFisher, San Jose, CA). Peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein databases with the acquired fragmentation pattern by the software program, SEQUEST (ver. 28). (ThermoFisher, San Jose, CA). Enzyme specificity was set to partially tryptic with 2 missed cleavages. Modifications included carboxyamidomethyl (cysteines, fixed) and oxidation (methionine, variable). Mass tolerance was set to 2.0 for precursor ions and 1.0 for fragment ions. The database searched was the Human IPI databases version 3.6. Because we used HEK293 cells the Human IPI database was used. The number of entries in the database was 160,900 which included both the target (forward) and the decoy (reversed) human sequences. Spectral matches were filtered to contain less than 1% FDR at the peptide level based on the target-decoy method. Finally, only tryptic matches were reported and spectral matches were manually examined. When peptides matched to multiple proteins, the peptide was assigned so that only the most logical protein was included (Occam's razor). This same principle was used for isoforms when present in the database.

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Thymic epithelial cells from WT and AB0/0 mouse model

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