br Specifications table br Value

Specifications table
Value of the data
Data
Experimental design, materials and methods
Fractionation of human plasma samples Frozen human plasma samples from healthy volunteers were obtained from expired stocks of local blood banks, pooled and fractionated by anion exchange chromatography (AXC). Briefly, 125mL of pooled human plasma were dialyzed against equilibration buffer (50mM Hepes pH 6, 60mM NaCl, 1mM EDTA) and then centrifuged at 10,000g for 30min at 4°C, diluted 3-fold in equilibration buffer and subjected to tandem filtration through Whatman 1 qualitative filter paper (Whatman, UK) and a 0.45µm membrane. The resulting sample was loaded at a flow rate of 10cm/h onto an XK-26 chromatography column (GE Healthcare, USA) packed with 10mL of DE-52 gel, which was next washed with equilibration buffer until absorbance at 280nm returned to baseline (bound protein represented, in average, 1.2% of the initial protein contents of the sample and no more than 60% of the total protein binding capacity estimated for DE-52 at pH 6.0). Then, the column was washed sequentially with preparations of equilibration buffer containing increasing concentrations of NaCl (0.3M, 0.6M and 1M) at 30cm/h, and the protein species still bound to the column afterwards were eluted by the successive application of 0.02M sodium acetate pH 3.0, water and 0.01M NaOH. Chromatography fractions were used either separately or pooled in a single sample (ELUAXC) [2] (Fig. 1). Upon collection, a protease inhibitor cocktail consisting of 1μg/mL leupeptin, 1μg/mL pepstatin A, 1μg/mL soybean trypsin inhibitor and 1mM PMSF was added to the eluted fractions, which were subsequently dialyzed against 10mM Hepes pH 7.0, 0.15M NaCl, 1mM CaCl2 and stored at −20°C until used.
Virus-binding activity of plasma fractions Microtiter plates were coated with 1µg protein content of the corresponding AXC chromatography fraction diluted in 50µL of 0.05M sodium carbonate/bicarbonate buffer, pH 9.6. After coating, the plates were washed with 0.05% Tween 20 in 10mM Hepes pH 7.0, 0.12M NaCl, 1mM CaCl2 (HBCa-T) and remaining non-specific ursolic acid were blocked with 1% Bovine Serum Albumin in HBCa-T for 1h at 37°C. Specified dilutions of mouse-derived DENV2 were added to the plates and the binding reaction was allowed to proceed for 2h at 37°C. Bound virus was detected by incubation with biotinylated Mab 4G2 at 5µg/mL (1h at 37°C) followed by washing with HBCa-T and incubation with a streptavidin–peroxidase conjugate (1h at 37°C). After washing, the plates were developed by adding 0.001% H2O2, 1mg/mL o-phenylenediamine in 0.01mol/L citric acid-phosphate buffer pH 5.0 for 20min at room temperature, stopping the reactions by adding 20µL of 2.5M H2SO4, and reading absorbance at 492nm in a microplate reader (Fig. 2).
Protein composition of AXC eluates The pH of the AXC eluates was first adjusted to 8.0 by adding 0.4M Tris–HCl pH 8.0, 3M urea (i.e. 0.3M NaCl, 0.6M NaCl, 1M NaCl, AcO−, H2O and NaOH). Disulfide bonds were reduced and the resulting free thiol groups were alkylated by adding dithiothreitol to a final concentration of 10mM and incubating samples at 37°C for 2h, and then adding acrylamide to a concentration of 25mM and incubating the samples for 1h at 25°C. The reduced/alkylated samples were diluted with water to 0.1M Tris/HCl pH 8.0, 0.75M urea and sequencing grade trypsin (Promega, USA) was added to an enzyme:substrate ratio of 1:50, incubating the resulting mixture for 16h at 37°C. Before analysis by LC-MS/MS, all peptide mixtures were desalted in Sep-Pak tC18 cartridges (Waters, USA), eluted in 60% acetonitrile, 0.1% formic acid, concentrated by evaporation under vacuum and dissolved in 0.1% formic acid. Peptide digest mixtures were analyzed by liquid chromatography coupled to MS experiments (LC-MS/MS), using an Ultimate 3000 RSLC nano system (Thermo Fisher Scientific, USA) connected on-line to a linear trap quadrupole (LTQ)-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). One microliter of each sample was injected into an Acclaim PepMap trap column (2cm×75μm i.d., C18, 3μm, 100Å) (Dionex, USA) at 5µL/min, using an aqueous solution containing 0.05% (v/v) trifluoroacetic acid and 1% acetonitrile. After three minutes, the trap column was connected to an Acclaim PepMap RSLC 15cm×75μm i.d., C18, 2μm, 100Å analytical column (Dionex, USA). The peptides were eluted using a linear 2–35% gradient (solvent A: 0.1% (v/v) formic acid in HPLC-grade water; solvent B: 0.1% (v/v) formic acid in HPLC-grade acetonitrile) at 300nL/min over 66min, followed by a washing step of 7min at 90% solvent B. MS spectra were acquired in the orbitrap analyzer over a mass range of 300–2000 Th. Peptide fragmentation was performed with the collision induced dissociation method at a normalized collision energy of 35%. MS/MS spectra were acquired in the linear trap.