MassIVE MSV000086649

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Mock-, SARS-CoV-2-, and UV-SARS-CoV-2-infected human lung microvascular endothelial cells (HL-mECs), at day 1, 2 and 3 p.i.

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Description

Human lung microvascular endothelial cells (HL-mECs) purchased from Lonza Clonetics (Walkersville, MD, USA) and cultured in EGM-2MV (Lonza) containing 10% (FBS). The proteomic analysis was performed on Mock-, SARS-CoV-2-, and UV-SARS-CoV-2-infected HL-mECs, collected at day 1, 2 and 3 p.i. For each condition, about 2 x 10^6 cells were used.Cells were lysed and proteins were extracted, reduced/alkylated and enzymatically digested using Easy PepTM Mini MS Sample Prep Kit (Thermo Fisher Scientific Rockford, IL, USA). Tryptic and Lys-C digestion was carried out on 100 microg and 70 microg of the two examined preparations, respectively. Following the kit protocol, in less than 3 h and for each examined condition, peptides were generated, cleaned-up to prepare detergent-free samples and resuspended in 0.1% formic acid (Sigma-Aldrich Inc., St. Louis, MO, USA) for LC-MS/MS analysis. Peptide mixtures were analyzed using Eksigent nanoLC-Ultra 2D System (Eksigent, part of AB SCIEX Dublin, CA, USA) configured in trap-elute mode. Briefly, samples (0.8microg injected) were first loaded on a trap (200 microm x 500 microm ChromXP C18-CL, 3 microm, 120 A) and washed with the loading pump running in isocratic mode with 0.1% formic acid in water for 10 min at a flow of 3 microL/min. The automatic switching of autosampler ten-port valve then eluted the trapped mixture on a nano reversed phase column (75 microm x 15 cm ChromXP C18-CL, 3 microm, 120 A) through a 133 min gradient of eluent B (eluent A, 0.1% formic acid in water; eluent B, 0.1% formic acid in acetonitrile) at a flow rate of 300 nL/min. In depth, gradient was: from 5-10% B in 3 min, 10-40% B in 110 min, 40-95% B in 12 min and holding at 95% B for 8 min. The eluted peptides were directly analyzed on a LTQ-OrbitrapXL mass spectrometer (Thermo Fisher Scientific) equipped with a nanospray ion source. The spray capillary voltage was set at 1.7 kV and the ion transfer capillary temperature was held at 220 C. Full MS spectra were recorded over a 400 - 1600 m/z range in positive ion mode, with a resolving power of 60000 (full width at half-maximum) and a scan rate of 2 spectra/s. This step was followed by five low-resolution MS/MS events that were sequentially generated in a data-dependent manner on the top five ions selected from the full MS spectrum (at 35% collision energy), using dynamic exclusion of 0.5 min for MS/MS analysis. Globally, 45 raw data files are uploaded (MOCK: Day 1, n=5, Day 2, n=5, Day 3, n=5; SARS-CoV-2: Day 1, n=5, Day 2, n=5, Day 3, n=5; UV-SARS-CoV-2: Day 1, n=5, Day 2, n=5, Day 3, n=5).

Keywords: SARS-CoV-2 ; Endothelial cells ; Lung ; UV

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