AP-MS analysis of m7GTP-binding proteins in Murine norovirus infected BV-2 cells (v2)
SILAC-labelling analysis of murine BV-2 cells infected at high multiplicity of infection (10 TCID50/cell) and harvested at 4h or 9h post-infection. Lysates were then suject to m7GTP-sepharose affinity purification to enrich for translation initiation factors. As norovirus translation uses a VPg protein covalently linked to the 5' of its RNAs for initiation factor recruitment it was hypothesised that norovirus infection could modify initiation factor complexes. A paired dataset investigates the effects of MNV infection on total protein levels in infected cells. This dataset is a Maxquant reanalysis of data previously submitted under PXD004019
Sample Processing Protocol
Cells were labelled in SILAC DMEM containing R0K0 (0h/Mock), R6K4 (4h post-infection) or R10K8 (9h post-infection). For one repeat the R6K4 and R10K8 labelled cells were swapped. m7G sepharose enrichment of cap-binding proteins was performed using γ-Aminophenyl-m7GTP (AC-1555, Jena Bioscience) as described previously (Chung et al., 2014). In brief, cells were lysed in m7G lysis buffer (1% Triton X-100, 100 mM KCl, 0.1 mM EDTA, 10% glycerol, 2 mM MgCl2, 20 mM Hepes. pH 7.6, supplemented with protease inhibitors). Lysates were incubated on ice for 5 min at 4 °C. Debris was removed by centrifugation at 15,000 × g for 5 min and sample concentration normalized by BCA assay (Pierce). The cleared lysates were subsequently incubated with prewashed m7GTP-Sepharose (50 μl bead volume; Jena Bioscience) for 2 h at 4°C. Unbound proteins were removed by centrifugation at 1000 × g for 5 min followed by washing 3 times using ice-cold lysis buffer lacking Triton X-100. Finally, bound proteins were eluted by boiling in SDS-PAGE reducing sample buffer and retained for downstream analysis. Samples were run on SDS-PAGE electrophoresis on precast gels. Gels were cut into 10 slices which were subjected to in-gel tryptic digestion using a ProGest automated digestion unit. The resulting peptides were 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 (Dionex). 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 (Dionex) 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 3min., 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 a stainless steel 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 2000 and the top six 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, 40%; activation q, 0.25; activation time 10ms; and minimum ion selection intensity, 500 counts.
Data Processing Protocol
The raw data files were processed and quantified using Maxquant v188.8.131.52 and searched against the Uniprot Mouse database (51,418 entries, dated May 14th, 2016) plus a custom fasta file generated in-house containing the MNV-1 (accession DQ285629) protein sequences using the built-in Andromeda search engine. Peptide 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 and N-terminal acetylation 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.
Emmott E, Sorgeloos F, Caddy SL, Vashist S, Sosnovtsev S, Lloyd R, Heesom K, Locker N, Goodfellow I. Norovirus-mediated modification of the translational landscape via virus and host-induced cleavage of translation initiation factors. Mol Cell Proteomics. 2017 Jan 13. pii: mcp.M116.062448 PubMed: 28087593