Metadata |
datasetIdentifier | PASS01114 |
datasetType | SRM |
submitter | Maria Loureiro <eugenialoureiro@yahoo.com.ar> |
submitter_organization | Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA. |
lab_head_full_name | Elina Zuniga |
lab_head_email | eizuniga@ucsd.edu |
lab_head_organization | Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA. |
lab_head_country | U.S.A. |
datasetTag | ArenavirusNP2017 |
datasetTitle | DDX3 Is Exploited By Arenaviruses To Suppress Type I Interferons And Favor Their Replication |
publicReleaseDate | 2017-10-17 00:00:00 |
finalizedDate | 2017-10-17 12:43:32 |
summary | Ananlysis of the arenavirus Nucleoprotein (NP) interactome in Human epithelial cells (ATCC® CCL185™) |
contributors | María Eugenia Loureiro, Andre Luiz Zorzetto-Fernandes, Sheli Radoshitzky, Xiaoli Chi, Simone Dallari, Nuha Marooki, Psylvia Lèger, Sabrina Foscaldi, Sonia Sharma, Nora López, Juan Carlos de la Torre, Sina Bavari and Elina Zuniga* |
publication | Loureiro ME, Zorzetto-Fernandes AL, Radoshitzky S, Chi X, Dallari S, Marooki N, Lèger P, Foscaldi S, Sharma S, López N, de la Torre JC, Bavari S and Zuniga E. (2017) DDX3 Is Exploited By Arenaviruses To Suppress Type I Interferons And Favor Their Replication. The EMBO Journal.Submitted |
growth | A549 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (11965-118, Gibco, Grand Island, NY, USA) supplemented with 2 mM L-glutamine (25030081, Thermo Scientific), 50 U/mL penicillin-streptomycin (15140-163, Gibco), plus 10% heat-inactivated FBS (Lonza). |
treatment | A549 cells were either transfected with a plasmid expressing Linfochoriomeningitis virus (LCMV) or Lassa Virus (LASV) Nucleoprotein tagged to HA peptide in its C-terminal end (NP-HA) (Martinez-Sobrido L et al (2007)J. Virol. 81: 12696-703
Alternatively, as negative controls, cells were trasnfected with a plasmid expressing an HA-tagged unrelated protein (HA-USP14: plasmid encoding ubiquitin-specific protease 14 fused to HA epitope to its N-terminal end. Sowa ME et al (2009) Cell 138: 389-403 ) or infected with a newly generated recombinant virus expressing HA-tagged GFP as foreign protein (3rLCMV-GFPHA) |
extraction | 24h post-trasnfection, cells were washed twice with PBS and lysed with 200 l/well of Immunoprecipitation lysis buffer (Pierce IP Lysis Buffer, Thermo Scientific), supplemented with Complete EDTA-Free Protease Inhibitor Cocktail tablet (04693159001, Roche Applied Science). When indicated, samples were treated with 100 μg/mL RNAseA for 20 minutes before co-immunoprecipitation. All lysates were cleared by centrifugation at 12.000 rpm for 30 min at 4 C. After protein quantification, lysates were incubated at a ratio of 1mg lysate/50μL of resin (mouse monoclonal anti-HA antibody (clone HA-7) conjugated to agarose beads, A2095, Sigma-Aldrich), rotating overnight at 4C. Beads were then washed 4 times with IP Lysis Buffer and 2 times with PBS. |
separation | Proteins were recovered with 200 ul of 250μg/mL HA-peptide (I2149, Sigma Aldrich) per 50 ul of resin. Proteins present in eluates were concentrated using Ultra-4, membrane PLGC Ultracel-PL (UFC801024, Amicon) and resuspended in TNE (50 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA) buffer. Samples were adjusted to 0.1% RapiGest SF reagent (Waters Corp.) and boiled for 5 min, followed by addition of TCEP (Tris (2-carboxyethyl) phosphine) to 1 mM final concentration and incubation at 37C for 30 min. |
digestion | Samples were adjusted to 0.1% RapiGest SF reagent (Waters Corp.) and boiled for 5 min, followed by addition of TCEP (Tris (2-carboxyethyl) phosphine) to 1 mM final concentration and incubation at 37C for 30 min. Samples were carboxymethylated with 0.5 mg/ml of iodoacetamide for 30 min at 37C, followed by neutralization with 2 mM TCEP, and digested with trypsin (trypsin:protein ratio - 1:50) overnight at 37C. RapiGest was degraded and removed by treatment with 250 mM HCl at 37C for 1 h, followed by centrifugation at 14,000 rpm for 30 min at 4C. |
acquisition | The soluble fraction was applied to a C18 desalting column (Thermo Scientific, PI-87782). Desalted peptides were eluted from the C18 column into the mass spectrometer using a linear gradient (5–80%) of ACN (Acetonitrile) at a flow rate of 250 μl/min for 1h. The buffers used to create the ACN gradient were: Buffer A (98% H2O, 2% ACN, 0.1% formic acid, and 0.005% TFA) and Buffer B (100% ACN, 0.1% formic acid, and 0.005% TFA). Analysis of desalted-peptides was performed by ultra high-pressure liquid chromatography (UPLC) coupled with tandem mass spectroscopy (LC-MS/MS) using nano-spray ionization. Nano-spray ionization was done using a TripleTof 5600 hybrid mass spectrometer (ABSCIEX) interfaced with nano-scale reversed-phase UPLC (Waters corporation nano ACQUITY) using a 20 cm-75 micron ID glass capillary packed with 2.5-µm C18 (130) CSHTM beads (Waters corporation). MS/MS data were acquired in a data-dependent manner in which the MS1 data was acquired for 250 ms at m/z of 400 to 1250 Da and the MS/MS data was acquired from m/z of 50 to 2,000 Da. The Independent data acquisition (IDA) parameters were as follows; MS1-TOF acquisition time of 250 milliseconds, followed by 50 MS2 events of 48 milliseconds acquisition time for each event. The threshold to trigger MS2 event was set to 150 counts when the ion had the charge state +2, +3 and +4. The ion exclusion time was set to 4 seconds. |
informatics | the collected data were analyzed using Protein Pilot 4.5 (ABSCIEX) for peptide identifications. Identified proteins were considered specific when at least two or more unique tryptic peptides were detected with a degree of confidence of 99% , and were never present in HA-USP14 or 3rLCMV-HA-GFP negative controls. Spectral count normalization (NSC) was used to estimate the relative protein abundance as described in (Paoletti et al (2006) PNAS 103: 18928-33). |
instruments | Analysis of desalted-peptides was performed by ultra high-pressure liquid chromatography (UPLC) coupled with tandem mass spectroscopy (LC-MS/MS) using nano-spray ionization. Nano-spray ionization was done using a TripleTof 5600 hybrid mass spectrometer (ABSCIEX) interfaced with nano-scale reversed-phase UPLC (Waters corporation nano ACQUITY) using a 20 cm-75 micron ID glass capillary packed with 2.5-µm C18 (130) CSHTM beads (Waters corporation). |
species | Human |
massModifications | None |