Phosphoproteomic characterization of infulenza A virus infected human macrophages
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture, Macrophage
DISEASE(S): Influenza
SUBMITTER: 
Tuula Nyman
LAB HEAD: Tuula Nyman
PROVIDER: PXD001620 | Pride | 2016-08-08
REPOSITORIES: Pride
Dataset's files
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Publications
Phosphoproteomics to Characterize Host Response During Influenza A Virus Infection of Human Macrophages.
Söderholm Sandra S Kainov Denis E DE Öhman Tiina T Denisova Oxana V OV Schepens Bert B Kulesskiy Evgeny E Imanishi Susumu Y SY Corthals Garry G Hintsanen Petteri P Aittokallio Tero T Saelens Xavier X Matikainen Sampsa S Nyman Tuula A TA
Molecular & cellular proteomics : MCP 20160802 10
Influenza A viruses cause infections in the human respiratory tract and give rise to annual seasonal outbreaks, as well as more rarely dreaded pandemics. Influenza A viruses become quickly resistant to the virus-directed antiviral treatments, which are the current main treatment options. A promising alternative approach is to target host cell factors that are exploited by influenza viruses. To this end, we characterized the phosphoproteome of influenza A virus infected primary human macrophages ...[more]
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Project description:Human primary macrophages were infected with influenza A virus (H3N2/Udorn strain, HA 256) for 6 hrs or left untreated. Cells were collected and lysed with HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, pH 7.4) including protease and phosphatase inhibitor cocktails. The cell lysates were centrifuged and the supernatant was collected and the protein content was measured with Bio-Rad DC™ protein assay (Bio-Rad). The proteins were reduced, alkylated, and enzymatically digested in-solution with trypsin. The digestion was stopped by adding FA (final c = 1 %). The samples were centrifuged 9168 x g for 10 min and desalted with Sep-Pak Vac RP C18 cartridges (Waters, MA, USA), following fractionation by strong cation exchange chromatography (SCX). The peptides were separated on a 200 x 4.6 mm, 5 ?m, 200 Å PolySULFOETHYL A™ column (PolyLC, USA). The fractions were vacuum centrifuged and desalted as before, following phosphopeptide-enrichment with PHOS-Select™ Iron Affinity Gel (Sigma Aldrich, MO, USA). LC-MS/MS was performed with a Q Exactive hybrid quadrupole-orbitrap tandem mass spectrometer coupled to an EASY-nLC 1000 nanoflow liquid chromatograph (Thermo Fisher Scientific). A 100 ?m x 3 cm trap column and a 75 ?m x 15 cm analytical column were in-house packed with Magic C18AQ resin (200 Å, 5 ?m; Michrom Bioresources). The mobile phases were 2% acetonitrile, 0.2% formic acid (A) and 95% acetonitrile, 0.2% formic acid (B). LC gradient elution condition was 2% B (0 min), 20% B (70 min), 40% B (100 min), and then 100% B (105-110 min), with a flow rate of 300 nl/min. Data dependent acquisition was performed in positive ion mode. MS spectra were acquired from m/z 300 to m/z 2000 at a resolution of 70,000 at m/z 200 with a target value of 1,000,000 and maximum injection time of 120 ms. The 10 most abundant precursor ions of which charge states were 2+ or higher were selected for higher energy collisional dissociation (HCD) with an isolation window of 2 and normalized collision energy of 30. MS/MS spectra were acquired at a resolution of 17,500 at m/z 200 with a target value of 50,000, maximum injection time of 250 ms, and the lowest mass fixed at m/z 100. Dynamic exclusion duration was 30 s.
2017-03-29 | MSV000080717 | MassIVE
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