SARS-CoV-2 spike analysis before and after UV-C treatment
Ontology highlight
ABSTRACT:
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).
2020-12-30 | MSV000086649 | MassIVE
Project description:Two hundred microliters per protein sample, collected at the end of isolation, were concentrated to 50 microL in a vacuum concentrator at 60C and treated with RapiGestTMSF reagent (Waters Co, Milford, MA, USA) at the final concentration of 0.25% (w/v). The resulting suspensions were incubated under stirring at 100C for 20 minutes. Subsequently the samples were cooled to room temperature and centrifuged 10 min at 2200 g. The protein concentration was assayed using the Invitrogen Qubit Protein BR Assay Kit (Life Technologies Corporation, Thermo Fisher, Eugene, ORE, USA) and 50 microg proteins from each sample were digested overnight at 37C by adding Sequencing grade Modified Trypsin (Promega Inc., Madison, WI, USA) at an enzyme substrate ratio of 1:50 (w/w) in 0.1M NH4HCO3 pH 7.9 buffer with 10% CH3CN. An additional aliquot of trypsin (1:100 w/w) was then added the next day, and the digestion continued for 4h. The enzymatic digestion was chemically stopped by acidification with 0.5%Trifluoroacetic Acid (TFA) (SigmaAldrich Inc., St. Louis, MO, USA), and a subsequent incubation at 37C for 45 minutes completed the RapiGest acid hydrolysis. Water immiscible degradation products were removed by centrifugation at 13,000 rpm for 10 minutes. Finally, the tryptic digest mixtures were desalted using PierceTM C 18 spin columns (Thermo Fisher Scientific,Pierce Biotechnology, Rockford, Il, USA), according to manufacturer protocol and were resuspended in 0.1% formic acid (Sigma-Aldrich Inc., St. Louis, MO, USA) in water (LC MS Ultra CHROMASOLV, Honeywell Riedelde HaenTM, Muskegon, MI, USA) at a concentration of 0.1 microg/microL.
The nanoLC MS/MS experiments were performed using the LTQ Orbitrap XL ETD mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with Eksigen nanoLC-Ultra 2D System (Eksigent, part of AB SCIEX Dublin, CA, USA) configured I 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 minutes at a flow of 3 microL/min. The automatic switching of auto-sampler 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 130 minutes 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 5 min, 10 40% B in 85min, 40 95% B in 27 min and holding at 95% B for 10 min. The eluated peptides were directly analyzed into a LTQ Orbitrap XL ETD through a nano electrospray ion source (Thermo Fisher Scientific); each sample was analyzed in at least 3 technical replicates. The spray capillary voltage was set at 1.7 kV and the ion transfer capillary temperature was held at 220C. Full MS spectra were recorded over a 400 1600 m/z range in positive ion mode, with a resolving power of 30000 (full width at half-maximum) and a scan rate of 2 spectra/s. This step was followed by 5 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. Mass spectrometer scan functions and high-performance liquid chromatography solvent gradients were controlled by the Xcalibur data system version 1.4 (Thermo Fisher Scientific, MA, USA).
File description:
HD > Healthy donors
LRPCa > low risk patients (bladder cancer)
HRPCa > high risk patients (bladder cancer)
Sub > subject
I, II, III etc...> technical replicates
2024-11-12 | MSV000096381 | MassIVE
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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Project description:Proteomics study of human urine exosomes. This data describes the proteomic complement of the human exosomal urine fraction from 10 healthy volunteers (5 male, 5 female) aged 23 to 36 years. 50 µg protein from each sample were fragmented on a 4%/12% SDS-polyacrylamide gel. Following staining, each gel track was separated into 28 equal sections, which were processed individually. Peptides were separated by reverse-phase chromatography (Dionex, Sunnyvale, CA) and LC-MS/MS was performed using an Eksigent NanoLC-1D Plus (Eksigent Technologies, Dublin, CA) HPLC system and an LTQ Orbitrap mass spectrometer (ThermoFisher, Waltham, MA). Peptides from each gel segment were analysed twice: (1) with a dynamic exclusion list and (2) a fixed exclusion list for the abundant protein uromodulin which was superimposed on a dynamic exclusion. MS data were processed using SEQUEST Bioworks Browser (version 3.3.1 SP1, ThermoFisher) to generate MS/MS peak lists. Combined peak list files were submitted to the MASCOT search algorithm (version 2.2.1, Matrix Science, London UK) and searched against the IPI-Human database.
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Project description:Non-coding RNA profiling by microarray in an Affymetrix® miRNA Array, version 4.1 (Santa Clara, CA, USA), commercially available by Thermo Fisher Scientific (Waltham, MA) We aimed to profile the expression of the entire population of annotated small non-coding RNAs in the Temporal Cortex (TC) of Alzheimer diseased (AD) individuals compared to age-matched controls.
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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).
2021-02-15 | MSV000086866 | MassIVE
Project description:The isolated Evs from urine sample (from healthy preschool children born late preterm (LP) and full-term (T)) were resuspended in 0.1 M ammonium bicarbonate, pH 7.9 and were trypsinized according to procedures previously reported, using 50 microg of proteins from each sample.
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2021-02-15 | MSV000086867 | MassIVE
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2020-06-29 | GSE143166 | GEO
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