MassIVE MSV000097027

Partial Public

Sus scrofa WT and DMD cardiac decellularized ECM

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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. [doi:10.25345/C5JM23T7T] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Biomaterials ; Extracellular matrix ; Duchenne Muscular Dystrophy ; Hydrogel ; 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
Pierluigi Mauri, Institute of Biomedical Technologies, Italy
Raffaello Vigano, Institute of Biomedical Technologies, Italy
Submitting User: Dds100
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