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SARS-CoV-2 spike analysis before and after UV-C treatment

ABSTRACT: The tryptic S peptide mixtures were analysed by by the Eksigent nanoLC-Ultra 2D System (Eksigent, AB SCIEX Dublin, CA, USA) configured in trap-elute mode as previously described(57) through a 70 min gradient of eluent B (eluent A, 0.1% formic acid in water; eluent B, 0.1% formic acid in acetonitrile). Briefly, 600 ng of digested S protein was loaded on a first trap (200-500 microm ChromXP C-18-CL, 3 microm, 120 Angstrom) and then eluted on a reverse-phase nano-column (75 microm x 15 cm ChromXP C18-CL, 3 microm, 120 Angstrom) at a flowrate of 300 nL/minute. The peptide mixtures were separated according to specific gradients (from 5-10% B in 2 min, 10-60% B in 38 min, 60-95% B in 8 min and holding in 95% B for 9 min before returning at 5% B in 3 min) and analysed by LTQ-OrbitrapXL mass spectrometer (Thermo Fisher Scientific, Waltham, CA, USA) equipped with a nanospray ion source which were managed by Xcalibur software version 1.4 (Thermo Fisher Scientific, Waltham, CA, USA). Full mass spectra were acquired at 400-1600 m/z scan range in positive ion mode and with a resolution setting of 60000 FWHM (m/z 200) and a scan rate of 2 spectra/second. The full MS 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), with an isolation window of 2 m/z from the survey scan.

INSTRUMENT(S): LTQ Orbitrap XL

ORGANISM(S): Sars Coronavirus (ncbitaxon:227859)

SUBMITTER: Pierluigi Mauri Dario Di Silvestre Letizia Bernardo

PROVIDER: MSV000090915 | MassIVE | Fri Dec 16 08:15:00 GMT 2022

REPOSITORIES: MassIVE

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Project 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).

Project description: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).

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SARS-CoV-2 spike analysis before and after UV-C treatment

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