Mass spectrometry analysis of DENV-2 Infected HeLa Supernatant

II.15 Supernatant harvest for HeLa stimulation experiments

HeLa were seeded at a density of 1x10⁶ cells in a 75 cm³ vented flask in complete media and incubated for 24 hr at 37 °C, 5% CO₂. Following incubation, cells had media removed and were either inoculated with DENV-2 prepared (MOI=1) in 5 mL DMEM or received 5 mL DMEM only, rocking every 15 min for 90 min. Inoculum was then removed and cells received 20 mL of complete media. Cells were left to incubate for 48 hr at 37 °C, 5% CO₂. Following incubation, cell culture media was harvested, and cells lysed in TRIzol for RT-qPCR to confirm DENV infection. Harvested supernatant was clarified by centrifugation at 2,600 x g for 5 minutes before storing at -80°C. Conditioned media is the term given to the supernatant of cells generated in the absence of DENV-infection.

II.16 Sized based fractionation of DENV-infected HeLa supernatant

Supernatants were either unfiltered or fractionated into large (>50 kDa) and small (<50 kDa) proteins by Ultra-15, MWCO 50 kDa centrifugal filters (Amicon) for 15 min at RT, at 2,600 x g. Supernatant fractions were then reconstituted to the starting volume (20 mL) in DMEM.

II.18 Serum free fractionated supernatant generation and mass spectrometry analysis

For mass spectrometry analysis, HeLa were infected in a 75 cm³ vented flask in the absence of FCS and then had supernatant protein quantitated by BioRad protein assay. 10 μg of protein from supernatants was prepared by Sera-Mag Carboxylate SpeedBeads (Cytiva) clean up as per manufacturer’s instructions to remove contaminating salts and detergents. Protein was then digested using 0.5 μg trypsin gold (Promega Corporation, Alexandria, NSW, Australia) overnight at 37 °C. Peptide clean-up was performed for further salt removal using Sera-Mag Carboxylate SpeedBeads which were then dissociated from peptides as per manufacturer’s instructions in preparation for analysis. Peptides were analysed by the Flinders Proteomics Facility, performed by Dr Alex Colella using Mass spectrometry (TSQ Altis™ Triple Quadrupole Mass Spectrometer, Thermo Scientific) as previously described by Sharma et. al (2009).²²³