MassIVE MSV000080717

Imported Reanalysis Dataset Public PXD001620

Phosphoproteomic characterization of infulenza A virus infected human macrophages

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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. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: primary human macrophages ; influenza A virus ; phosphorylation

Contact

Principal Investigators:
(in alphabetical order) 		Tuula Nyman, Institute of Biotechnology, University of Helsinki, N/A
Submitting User: ccms

Publications

Söderholm S, Kainov DE, Öhman T, Denisova OV, Schepens B, Kulesskiy E, Imanishi SY, Corthals G, Hintsanen P, Aittokallio T, Saelens X, Matikainen S, Nyman TA.
Phosphoproteomics to Characterize Host Response During Influenza A Virus Infection of Human Macrophages.
Mol. Cell Proteomics. 2016 Oct;15(10):3203-3219. Epub 2016 Aug 2.

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