Phosphoproteomic analysis of Bluetongue virus infection
Bluetongue virus causes infections in wild and domesticated ruminants, with the potential for causing significant morbidity, mortality and economic harm in both the developing and developed world. While vaccines have been developed, no treatment for acute infections exists. In this regard, a possible approach is to determine host-cell factors utilised by Bluetongue virus. We thus proceeded to characterize the phosphoproteome of BTV infected HeLa cells to elucidate the intracellular signalling pathways and identify critical host factors activated during Bluetongue virus infection.
Sample Processing Protocol
HeLa cells were SILAC labelled using SILAC medium (R0K0, R6K4, R10K8) from Dundee Cell Products for 8 cell passages. Cells were infected with an MOI=5. At 12 hours and 18 hours post infection, cells were washed with PBS and cells lysed using lysis buffer (8M Urea, 75mM NaCl, 50mM Tris, pH 8.2, Protease inhibitor cocktail (confirm supplier), 1mM NaF, 1mM Beta-glycerophosphate, 1mM orthovanadate, 10mM sodium pyrophosphate and 1mM PMSF). Cellular detritus was spun down and lysates combined in a 1:1:1 ratio prior to submission for phosphopeptide enrichment and analysis via mass spectrometry (University of Bristol). SILAC pools were reduced (20mM TCEP for 1hr at 55°C), alkylated (35mM iodoacetamide for 30min at room temperature) and proteins precipitated using 6 volumes of acetone (15h, -20°C). Precipitated proteins were resuspeded in 100mM TEAB to which 2.5% (w/w) trypsin was added and the samples incubated overnight at 37°C. Following digestion, the samples were resuspended in 5% formic acid and desalted using SepPak cartridges according to the manufacturer’s instructions (Waters, Milford, Massachusetts, USA)). Eluate from the SepPak cartridge was evaporated to dryness and resuspended for either TiO2-based or Fe-NTA-based phosphopeptide enrichment according to the manufacturer’s instructions (Pierce). Enriched phosphopeptides were then fractionated using a Dionex Ultimate 3000 nanoHPLC system in line with an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). In brief, peptides in 1% (vol/vol) formic acid were injected onto an Acclaim PepMap C18 nano-trap column (Thermo Scientific). After washing with 0.5% (vol/vol) acetonitrile 0.1% (vol/vol) formic acid peptides were resolved on a 250 mm × 75 μm Acclaim PepMap C18 reverse phase analytical column (Thermo Scientific) over a 150 min organic gradient, using 7 gradient segments (1-6% solvent B over 1min., 6-15% B over 58min., 15-32%B over 58min., 32-40%B over 5min., 40-90%B over 1min., held at 90%B for 6min and then reduced to 1%B over 1min.) with a flow rate of 300 nl min−1. Solvent A was 0.1% formic acid and Solvent B was aqueous 80% acetonitrile in 0.1% formic acid. Peptides were ionized by nano-electrospray ionization at 2.1 kV using an emitter with an internal diameter of 30 μm (Thermo Scientific) and a capillary temperature of 250°C. Tandem mass spectra were acquired using an LTQ- Orbitrap Velos mass spectrometer controlled by Xcalibur 2.1 software (Thermo Scientific) and operated in data-dependent acquisition mode. The Orbitrap was set to analyze the survey scans at 60,000 resolution (at m/z 400) in the mass range m/z 300 to 1800 and the top twelve multiply charged ions in each duty cycle selected for MS/MS in the LTQ linear ion trap. Charge state filtering, where unassigned precursor ions were not selected for fragmentation, and dynamic exclusion (repeat count, 1; repeat duration, 30s; exclusion list size, 500) were used. Fragmentation conditions in the LTQ were as follows: normalized collision energy, 35%; activation q, 0.25; activation time 10ms; and minimum ion selection intensity, 500 counts. Multistage activation was enabled with neutral loss masses of 32.7, 49 and 98.
Data Processing Protocol
The raw data files were processed and quantified using Maxquant v184.108.40.206 and searched against the Uniprot Human database (70,550 entries, downloaded September 19th, 2016) plus a custom fasta file generated in-house containing the BTV protein sequences (South African reference strain, Genbank accession numbers FJ969719-FJ969728). Precursor mass tolerance was set at 4.5ppm, and MS/MS tolerance was set at 0.5Da. Search criteria included carbaminomethylation of cysteine as a fixed modification. Oxidation of methionine, N-terminal acetylation, and STY phosphorylation were selected as variable modifications. Quantification was based on Light (Arg 0, Lys 0) Medium (Arg 6, Lys 4) and Heavy (Arg 10, Lys 8) SILAC labels. Searches were performed with full tryptic digestion, a minimum peptide length of 7 amino acids, and a maximum of 2 missed cleavages were allowed. The reverse database search option was enabled and the maximum false discovery rate for both peptide and protein identifications was set to 0.01. Quantitation was performed using a mass precision of 2ppm and the requantify option in Maxquant was enabled. The presented protein ratios represent the median of the raw measured peptide ratios for each protein.