MassIVE MSV000097631

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Proteomic Analysis of Mus musculus Heart Tissues WT and MDX treated (and not) with human CSPG4 CAR T

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Description

Mouse hearts were thawed and weighed into conical bottom glass vials; 2.5 ul of Universal Nuclease (ThermoFisher Scientific, Waltham, MA, USA) and 1 ml of Lysis Solution (ThermoFisher Scientific, Waltham, MA, USA) were added to each sample. Hearth samples were all mechanically homogenized in order to disrupt tissue; homogenized tissues were then centrifuged at 13.000 rpm for 10 minutes. Each supernatant was recovered, and the protein content was evaluated by using the Qubit protein assay performed with Qubit4 fluorimeter (Invitrogen, part of ThermoFisher Scientific, Waltham, MA, USA). 100 ug of proteins for each sample were reduced, alkylated, digested with Tryp/LysC enzymes and, finally, cleaned up according to the protocol of the EasyPep Mini MS sample preparation kit (ThermoFisher Scientific, Waltham, MA, USA). Each digested sample was dried after clean-up procedure and reconstituted in 0.1% formic acid (Sigma-Aldrich Inc., St. Louis, MO, USA) at a concentration of 1 ug/ul; samples were then diluted at 0.1 ug/ul before the nLC hrMS/MS analyses. Digested samples were separated using the Vanquish Neo UHPLC System (ThermoFisher Scientific, San Jose, CA, USA); this chromatographical system was implemented with PepMap Neo trap column (300 um X 5 mm, Thermo Scientific) and EASY Spray PepMap Neo Column (75 um I.D, 5 um, 15 cm; ThermoFisher Scientific, San Jose, CA, USA) for peptide separation, working in trap-and-elute mode (Flush Direction of trap column: forward). The Vanquish autosampler parameters were: temperature set at 7 C, loading volume of 4 ul; loading flow rate of 50 ul/min. For HPLC, following solvents were used: eluent A, H2O with 0.1% FA and eluent B, 80/20 ACN/H2O (%, v/v) with 0.1% FA. A 90-minute gradient (excluding ca. 1.4 min. of sample pickup and sample loading) was used for separation phase set as follows: 4% to 5% B in 1 min., 5% to 29% B in 54 min., 29% to 50% B in 34 min. and 50 to 70% B in 1 min. The column wash phase was from 90 to 96 min. at 99% B. The flow rate during the gradient was set at 300 nL/min. Mass spectra of heart samples were acquired in two technical replicates using a ThermoFisher Scientific Orbitrap Exploris 240 Mass Spectrometer for high-throughput bottom up proteomic profiling. The Orbitrap Exploris 240 mass spectrometer was equipped with the NSI Easy Spray source (Thermo Fisher Scientific, San Jose, CA, USA) working in a positive ion mode (positive ion spray voltage static at 1900V, ion transfer tube temperature 280 C, 35 C). [doi:10.25345/C5599ZD8D] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Duchenne ; CAR-T Cells ; Cardiac Tissue ; DatasetType:Proteomics

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Principal Investigators:
(in alphabetical order) 		Dario Di Silvestre, Institute of Biomedical Technologies, Italy
Francesca Brambilla, Institute of Biomedical Technologies, Italy
Raffaello Vigano, Institute of Biomedical Technologies, Italy
Submitting User: raffaellovigano
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