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

ABSTRACT: 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).

INSTRUMENT(S): Orbitrap Exploris 240

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

SUBMITTER: Francesca Brambilla Raffaello Vigano Dario Di Silvestre

PROVIDER: MSV000097631 | MassIVE | Tue Apr 15 08:36:00 BST 2025

REPOSITORIES: MassIVE

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Project description:Cardiac tissue decellularization was conducted on wild-type (WT) and dystrophic (DMD) pig heart tissues, derived from 1-day (1D) and 4-month-old (4M) animals. Briefly, the tissues were cut into pieces approximately 1 mm thick. The minced heart tissue was stirred in 0.3% SDS in a Milli-Q water for 48 h followed by treatment with a 3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA; X100-500ML) solution for a further 48 h. The decellularized extracellular matrices were washed using Milli-Q water for at least 3 days, lyophilized, and stored at -80C. Samples have been prepared to nanoscale liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) analysis with the MS compatible detergent RapiGest SF Surfactant (Waters Corporation, Milford, MA, USA; 186001861). Briefly, after recovering samples from -80C freezer, they were centrifuged at 13.000 rpm, for 10 min and the supernatant was discarded. Pellets were resuspended in 0.1 M Ammonium bicarbonate (NH4HCO3), pH=7.9 and mechanically homogenized. RapiGest SF was added to each sample to reach a final concentration of 0.2% v/v and samples were heated at 95C for 20 min. After centrifugation at 13.000 rpm for 10 min, samples were quantified using Qubit Protein Assay Kit on a QubitTM4 Fluorometer (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA); 50 microg of proteins were recovered from each sample and digested overnight with trypsin (Trypsin Gold, Promega, Madison, WI, USA; V5280) 1:50 enzyme/substrate ratio. Tryptic digestion was stopped with trifluoroacetic acid (TFA) to reach a final concentration of 0.5% v/v and samples were centrifuged (13.000 rpm for 10 min) to remove any matrix debris. Eventually, resulting peptides have been desalted and enriched with PepClean C18 Spin Columns (ThermoFisher Scientific, Waltham, MA, USA), according to the manufacturer s instructions. Each sample was analysed in two technical replicates on LC-MS/MS platform: Eksigent nanoLC-Ultra 2D System (Eksigent, Dublin, CA, USA) for nano liquid chromatography coupled with LTQ Orbitrap XLTM (Thermo Fisher Scientific, San Jose, CA, USA) for MS/MS analyses. In particular, peptides were separated with the following eluent gradient: (A) 0.1% formic acid in water; (B) 0.1% formic acid in acetonitrile; the gradient profile was 10-50% B in 104 min, 50-95% B in 17 min; 95% B for 9 min.

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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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