Computational protocol: Subgenomic promoter recognition by the norovirus RNA-dependent RNA polymerases

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Protocol publication

[…] RdRp crosslinking reactions were performed in clear-bottom 96-well microplates. The reactions were of 25 μl in volume and contained 2 μM purified MNV RdRp and 4 μM of 4SU-labeled RNA in a buffer containing 20 mM HEPES pH 7.5, 4 mM MgCl2, 1 mM MnCl2 and 1 mM DTT. The samples were transferred to microplates on ice and ultraviolet (UV) crosslinking was performed at 365 nm for 10 min. Trypsin digestion and precipitation of the peptide–RNA complexes were as described above. The pellet was suspended in a buffer containing 50 mM Tris pH 7.5, 200 mM NaCl and digested with RNAse A (1 μg) at 37°C for 30 min. Peptides and peptide–nucleotide conjugates were purified using a C18 column and analyzed by LC–ESI–MS/MS using an nanoliquid chromatography system (AB SCIEX) interfaced to an externally calibrated LTQ Orbitrap hybrid mass spectrometer equipped with a nanospray ion source (Thermo Scientific). A 4 μl aliquot of the peptide digest dissolved in 12 μl of mobile phase A was washed with buffer A for 5 min at a flow rate of 5 μl min−1 using a reversed-phase C18 trap column made from a 15 mm × 100 μm integrafrit column and packed with Magic C18 resin (200 Å pore size, 5 μm particle size; Bruker-Michrom). The desalted peptides were then separated with Buffer B gradient using a reversed-phase C18 column (Magic C18, 100 Å, 5 μm particle size, Bruker-Michrom) packed in a 150 mm × 75 μm, 15 μm tip (New Objective) and analyzed in positive ion mode. Buffer A consisted of 0.1% formic acid in acetonitrile:water (3%:97%, v/v) and Buffer B consisted of 0.1% formic acid in acetonitrile:water (97%:3%, v/v). The flow rate was 300 nl min−1 with the following gradient: 3% B from 0–1 min, ramp to 10% B from 1–9 min, ramp to 30% B from 9–28 min, ramp to 40% B from 28–31 min, ramp to 80% B from 31–35 min, hold at 90% B from 36–43 min, ramp to 3% B from 43–44 min, then re-equilibrate the column at 3% B for 14 min (58 min total run time). The mass spectrometer was operated in an automated data dependent mode, alternating between an FT-MS scan in the Orbitrap and five collision-induced dissociation (CID) scans in the linear ion trap. The Orbitrap monitored precursor ions from m/z 300 to m/z 2000 at 15 000 resolving power. The five most intense precursor ions in each scan were sequentially isolated in the linear trap with a 2 m/z isolation width and activated at 35% normalized collision energy. The cycle was continuously repeated throughout the entire separation with the dynamic exclusion set to 45 s with a repeat count of 2. Identification of peptides covalently attached to 4SU was performed using the Proteome Discoverer™ software by searching the raw LC–MS/MS data using SEQUEST® against the protein sequence and allowing for variable modifications corresponding to possible 4SU structures attached to the protein. […]

Pipeline specifications

Software tools Proteome Discoverer, Comet
Application MS-based untargeted proteomics
Organisms Mus musculus, Homo sapiens