Dataset of proteins mapped on HepG2 cells and those differentially abundant after expression of the dengue non-structural 1 protein

The data supplied in this article are related to the research article entitled “The effect of the dengue non-structural 1 protein expression over the HepG2 cell proteins in a proteomic approach” (K. Rabelo, M.R. Trugillo, S.M. Costa, B.A. Pereira, O.C. Moreira, A.T. Ferreira et al., 2016) [1]. The present article provides the inventory of peptides and proteins mapped in a hepatocyte cell line (HepG2) by mass spectrometry in the presence of the non-structural protein 1 (NS1) of Dengue 2 virus (DENV2). Cells were transfected with pcENS1 plasmid, which encodes the DENV2 NS1 protein, or the controls pcDNA3 (negative control) or pMAXGFP, encoding the green fluorescent protein (GFP), a protein unrelated to dengue. Differentially abundant protein lists were obtained by comparing cells transfected with pcENS1 and controls.

green fluorescent protein (GFP), a protein unrelated to dengue. Differentially abundant protein lists were obtained by comparing cells transfected with pcENS1 and controls.
& 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Subject area
Biology More specific subject area

Proteomics, Virology
Type of data  The data open new perspectives to identify the molecular mechanisms involving DENV NS1 protein in infected cells.

Data
HepG2 cells were transfected with the plasmids: pcENS1, pcDNA3 and pMAXGFP. To produce accurate data, we used three independent experimental biological replicates and samples were submitted to LTQ-Orbitrap XL (Thermo Scientific). Data analysis, using the PatternLab for Proteomics software, identified 14,138 peptides which mapped to 4756 proteins, from all conditions (HepG2 transfected with the three different plasmids and non-transfected cells) (Supplementary Table S1ah). Applying the maximum parsimony principle we found 2314 proteins (Supplementary Table S1g). Using the Tfold module we generate the differential abundance distribution when comparing: nontransfected HepG2 x cell transfected with pcDNA3 (Table 1); HepG2 transfected with pcDNA3 x pcENS1 ( Table 2) and cells transfected with pMAGFP x pcENS1 (Table 3) [1].

Plasmids
The recombinant plasmid pcENS1 was previously constructed in our laboratory [2], using the pcDNA3 mammalian expression vector (Invitrogen). It contains the sequence of 63 nucleotides that encodes 21 amino acids from the C-terminal portion of the DENV2 envelope (E) protein and the full length DENV2 ns1 gene. The vector pcDNA3 was used as a negative control, while the plasmid pMAXGFP (Amaxa), which encodes the green fluorescent protein (GFP) from Pontellina plumata copepod, was used as a control for expression of a DENV non-related protein.

Transfection
Transfection was performed by nucleofection with the Nucleofector V™ kit (Amaxa), according to manufacturer's recommendation. Briefly, HepG2 cells were seeded on 75 cm 2 bottles, harvested after 4 or 5 days with the aid of cell scrapes in 3 ml of CMF solution (8 g/L of NaCl; 0.4 g/L of KCl; 0.1 g/L of Na 2 SO 4 ; 0.39 g/L of Na 2 HPO 4 .12H 2 O; 0.15 g/L of KH 2 PO 4 ; 1.1 g/L of glucose; 0.0025 g/L of phenol red, pH 7.4), centrifuged at 500 g for 5 min and suspended in the nucleofection solution (Amaxa). Cell suspension with 5 μg of DNA plasmids (10 6 cells/100 μl/cuvettes) was submitted to an electric shock in the Nucleofector 6 equipment (Amaxa), using the T-28 program. Nine cuvettes were used for each sample (pcDNA3, pcENS1 or pMAXGFP). After shock, cells received 500 μL of DMEM with 10% FBS and were immediately transferred to microcentrifuge tubes containing another 500 μL DMEM with 10% FBS. Cells were seeded on 25 cm 2 flasks, incubated in humid atmosphere with 5% CO 2 at 37°C for 24 h.

Proteomic sample preparation
Cells were centrifuged at 500 g for 10 min and suspended in 50 mM ammonium bicarbonate buffer containing 0.2% of RapiGest™ SF (Waters). The protein concentration was determined using Qubit 2.0 s kit (Invitrogen) following the manufacturer's instructions. A total of 50 μg protein was used for each sample. Samples were treated with 5 μL of 100 mM dithiothreitol for reduction, incubated for 3 h at 37°C. After reaching room temperature, samples were alkylated with 5 μL of 400 mM iodoacetamide for 15 min, in the dark. Trypsin (Promega) was added in the ratio 1:50 enzyme/substrate and digestion was performed for 20 h, at 37°C. The reaction was stopped after adding formic acid to final concentration of 1%. Aliquots from this digestion were desalted by using POROS R2 C8-18 resin (Invitrogen), packaged in micropipette tips (Millipore) and equilibrated in TFA 1%. After washing with 0.1% TFA, peptides were eluted in 0.1% TFA with 70% acetonitrile and completely dried in the vacuum centrifuge.

Isoelectric focalization of peptides (OFFGEL)
Twenty five micrograms of peptides were solubilized in 1.8 mL of 0.01% ampholytes (OFFGEL buffer pH 3-10) containing 4% (v/v) glycerol and was submitted to the 3100 OFFGEL Fractionator with the OFFGEL Low Res Kit pH 3-10 (Agilent Technologies) immobilized pH gradient (IPG) DryStrips, following the Agilent's instructions. The peptides were also separated according to the manufacturer's instruction and optimized as described in Hubner et al. [3]. Twelve well fractionations were focused for 20 kV with a maximum current of 50 mA and power of 200 mW for 24 h. Each fraction was separately desalted as previously described and suspended in 40 μL of 1% formic acid. All fractions were analyzed on 10 cm reversed phase (RP) column coupled to an LTQ-Orbitrap XL mass spectrometer.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis
Desalted peptides fractions were loaded separately onto a 10 cm RP column coupled to the mass spectrometer by using a Proxeon easy-nLC-System (Thermo Scientific Easy-nLC II). Four microliters were initially applied to a 2 cm long (100 mm internal diameter) trap column packed with 5 mm, 200 Â e Magic C18 AQ matrix (Michrom Bioresources) followed by separation on a 10 cm long (75 mm internal diameter) separation column packed with the same matrix directly on a self-pack 5-15 mm Tip empty column (New Objective). Samples were loaded onto the trap column at 2 μL/min while chromatographic separation occurred at 200 nL/min. Mobile phase A consisted of 0.1% formic acid in water while mobile phase B consisted of 0.1% formic acid in acetonitrile. Peptides were eluted with a gradient of 2-40% of B over 32 min followed by up to 80% B in 4 min, maintaining at this concentration for 2 min more, before column equilibration. The HPLC system was coupled to the LTQ-Orbitrap XL via a nanoscale LC interface (Thermo Scientific). Source voltage was set to 1.9 kV, the temperature of heated capillary was set to 200°C and tube lens voltage to 48 and 100 V, respectively. The target precursor specters were acquired in ion trap full scan MS with 60,000 while FWHM full AGC target was set to 500,000. MS1 spectra were acquired on the Orbitrap analyzer (300-1700 m/z) at a 60,000 resolution (for m/z 445.1200). For each spectrum, the 10 most intense ions were submitted to CID fragmentation (minimum signal required of 10,000; isolation width of 2.5; normalized collision energy of 35.0; activation Q of 0.25 and activation time of 30 s, followed by MS2 acquisition on the linear trap quadrupole analyzer. Dynamic exclusion option was enabled and set with the following values for each parameter: repeat count ¼1; repeat duration¼30 s; exclusion list size ¼500; exclusion duration ¼45 s and exclusion mass width ¼10 ppm. Data were acquired in technical triplicates using the Xcalibur software (version 2.0.7).