Computational protocol: Heat Shock Protein 70 Family Members Interact with Crimean-Congo Hemorrhagic Fever Virus and Hazara Virus Nucleocapsid Proteins and Perform a Functional Role in the Nairovirus Replication Cycle

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[…] Quantification was performed with MaxQuant version () based on the two-dimensional centroid of the isotope clusters within each SILAC pair. The generation of values corresponding to peak list, SILAC-based, and extracted ion current-based quantitation, calculated posterior error probability, false-discovery rate based on search engine results, peptide-to-protein group assembly, and data filtration and presentation was carried out using MaxQuant. The derived peak list was searched with the Mascot search engine (Matrix Science, London, United Kingdom) against a concatenated database from the International Protein Index (IPI) human protein database and the reversed sequences of all proteins. Combined data sets from the two different experiments were deposited in the PRoteomics IDEntifications database (PRIDE) using the PRIDE converter tool and are presented as an annotated table (see Data set S1 in the supplemental material). [...] Peptide mixtures (2 μl) were analyzed by on-line nanoflow liquid chromatography using an nanoACQUITY-nLC system (Waters MS Technologies) coupled to an LTQ-Orbitrap Velos (Thermo Fisher Scientific) mass spectrometer equipped with a nanospray ion source. The analytical column (nanoACQUITY ultraperformance LC [UPLC] BEH130 C18) (15 cm by 75 μm, 1.7 μm capillary column) was maintained at 35°C with a flow rate of 300 nl/min. The gradient consisted of 3% to 40% acetonitrile–0.1% formic acid for 150 min followed by a ramp of 40% to 85% acetonitrile–0.1% formic acid for 5 min. Full-scan MS spectra (m/z range, 300 to 2,000) were acquired by the Orbitrap at a resolution of 30,000. The top 20 most intense ions from the MS1 scan (full MS) were selected for tandem MS by collision-induced dissociation, and all product spectra were acquired in the LTQ ion trap. Spectral data were transformed to .mgf files with Proteowizard (version 3.0) () and used for peptide identification performed with the Mascot (version 2.3.02; Matrix Science) search engine. Tandem MS data were searched against the predicted HAZV and human proteomes (UniProt release 2014_02). Mascot search parameters were as follows: precursor mass tolerance set to 10 ppm and fragment mass tolerance set to 0.8 Da. A maximum of one missed tryptic cleavage was permitted. Carbamidomethylation (cysteine) was set as a fixed modification and oxidation (methionine) as a variable modification. Mascot search results were further processed using Percolator. The false-discovery rate was <1%. Individual ion scores of >13 indicated identity or extensive homology (P < 0.05). Approximate label-free spectrum-counting-based quantitation of the proteins was determined using the exponentially modified protein abundance index (emPAI) calculated automatically by the Mascot search engine (). […]

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