PXD001620

PXD001620 is an original dataset announced via ProteomeXchange.

Title	Phosphoproteomic characterization of infulenza A virus infected human macrophages
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 μm, 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 μm x 3 cm trap column and a 75 μm x 15 cm analytical column were in-house packed with Magic C18AQ resin (200 Å, 5 μm; 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.
HostingRepository	PRIDE
AnnounceDate	2016-08-08
AnnouncementXML	Submission_2016-08-08_08:57:54.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Tuula Nyman
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue; monohydroxylated residue; acetylated residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2014-12-16 05:43:12	ID requested
⏵ 1	2016-08-08 08:57:55	announced

S, ö, derholm S, Kainov DE, Ö, hman 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, 15(10):3203-3219(2016) [pubmed]

curator keyword: Biomedical

submitter keyword: primary human macrophages, influenza A virus, phosphorylation

Tuula Nyman
contact affiliation	Institute of Biotechnology, University of Helsinki
contact email	tuula.nyman@helsinki.fi
lab head
Tuula Nyman
contact affiliation	Institute of Biotechnology
contact email	tuula.nyman@helsinki.fi
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD001620
     1. Label: PRIDE project
     2. Name: Phosphoproteomic characterization of infulenza A virus infected human macrophages