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Metadata
datasetIdentifierPASS01114
datasetTypeSRM
submitterMaria Loureiro <eugenialoureiro@yahoo.com.ar>
submitter_organizationDivision of Biological Sciences, University of California San Diego, La Jolla, CA, USA.
lab_head_full_nameElina Zuniga
lab_head_emaileizuniga@ucsd.edu
lab_head_organizationDivision of Biological Sciences, University of California San Diego, La Jolla, CA, USA.
lab_head_countryU.S.A.
datasetTagArenavirusNP2017
datasetTitleDDX3 Is Exploited By Arenaviruses To Suppress Type I Interferons And Favor Their Replication
publicReleaseDate2017-10-17 00:00:00
finalizedDate2017-10-17 12:43:32
summaryAnanlysis of the arenavirus Nucleoprotein (NP) interactome in Human epithelial cells (ATCC® CCL185™)
contributorsMaría 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*
publicationLoureiro 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
growthA549 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).
treatmentA549 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)
extraction24h 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 4C. Beads were then washed 4 times with IP Lysis Buffer and 2 times with PBS.
separationProteins 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 37C for 30 min.
digestionSamples 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.
acquisitionThe 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.
informaticsthe 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).
instrumentsAnalysis 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).
speciesHuman
massModificationsNone

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Listing of files:

 2.5M Oct 17  2017 3rLCMVGFPHA13.group
  45M Oct 17  2017 3rLCMVGFPHA13.wiff
 434M Oct 17  2017 3rLCMVGFPHA13.wiff.scan
  46K Oct 17  2017 3rLCMVGFPHA13_PeptideSummary.txt
  11K Oct 17  2017 3rLCMVGFPHA13_ProteinSummary.txt
 383K Oct 17  2017 3rLCMVGFPHA13__FDR.xlsx
 419M Oct 17  2017 HAUSP141416.wiff.scan
  31M Oct 17  2017 HAUSP1416.group
  47M Oct 17  2017 HAUSP1416.wiff
  40K Oct 17  2017 HAUSP1416_PeptideSummary.txt
 7.9K Oct 17  2017 HAUSP1416_ProteinSummary.txt
 339K Oct 17  2017 HAUSP1416__FDR.xlsx
  38M Oct 17  2017 LASVNPHA12.group
  46M Oct 17  2017 LASVNPHA12.wiff
 446M Oct 17  2017 LASVNPHA12.wiff.scan
 290K Oct 17  2017 LASVNPHA12_PeptideSummary.txt
  23K Oct 17  2017 LASVNPHA12_ProteinSummary.txt
 519K Oct 17  2017 LASVNPHA12__FDR.xlsx
  40M Oct 17  2017 LASVNPHA15.group
  47M Oct 17  2017 LASVNPHA15.wiff
 221M Oct 17  2017 LASVNPHA15.wiff.scan
 100K Oct 17  2017 LASVNPHA15_PeptideSummary.txt
  11K Oct 17  2017 LASVNPHA15_ProteinSummary.txt
 448K Oct 17  2017 LASVNPHA15__FDR.xlsx
  44M Oct 17  2017 LCMVNPHA 11.wiff
  45M Oct 17  2017 LCMVNPHA11.group
  26M Oct 17  2017 LCMVNPHA11.wiff.scan
 237K Oct 17  2017 LCMVNPHA11_PeptideSummary.txt
  30K Oct 17  2017 LCMVNPHA11_ProteinSummary.txt
 496K Oct 17  2017 LCMVNPHA11__FDR.xlsx
  32M Oct 17  2017 LCMVNPHA14.group
  47M Oct 17  2017 LCMVNPHA14.wiff
 168M Oct 17  2017 LCMVNPHA14.wiff.scan
  48K Oct 17  2017 LCMVNPHA14_PeptideSummary.txt
 4.3K Oct 17  2017 LCMVNPHA14_ProteinSummary.txt
 446K Oct 17  2017 LCMVNPHA14__FDR.xlsx
 5.9K Oct 17  2017 PASS01114_DESCRIPTION-2017-09-17_074904.txt
 5.9K Oct 17  2017 PASS01114_DESCRIPTION.txt
  21M Oct 17  2017 Reference search. human_LCMC_genome_NCBI (1)

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