Data from: Rotavirus NSP1 subverts the antiviral oligoadenylate synthetase-RNase L pathway by inducing RNase L degradation
Data files
May 25, 2023 version files 146.81 KB
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F20211015_Dai_Patton_Proteins.xlsx
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README.md
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Abstract
Methods
General methods are provided in
Detailed methods for analyzing the interactome of NSP1 are provided below.
Protein digestion
Individual samples containing proteins bound to magnetic beads were denatured in 8 M urea in 100 mM ammonium bicarbonate. Samples were incubated for 45 min at 57 °C with 10 mM Tris(2-carboxyethyl)phosphine hydrochloride to reduce cysteine residue side chains. These side chains were then alkylated with 20 mM iodoacetamide for one hour in the dark at 21 °C. The urea was diluted to 1 M urea using 100 mM ammonium bicarbonate. Trypsin was added at 1:100 w/w and the samples were placed on a rotator and digested for 14 hours at 37 °C.
Mass spectrometry (MS)
The beads were spun down an immobilized against the wall of the microcentrifuge tube using a magnet. The supernatant was removed and desalted using ZipTip pipette tips (EMD Millipore), dried down and resuspended in 0.1% formic acid. Peptides were analyzed by LC-MS on an Orbitrap Fusion Lumos equipped with an Easy NanoLC1200. Buffer A was 0.1% formic acid in water. Buffer B was 0.1% formic acid in 80% acetonitrile. Peptides were separated on a one-hour gradient from 0% B to 35% B. Peptides were fragmented by HCD. Precursor ions were measured in the Orbitrap with a resolution of 120,000. Fragment ions were measured in the Orbitrap with a resolution of 60,000.
MS data analysis
Data were analyzed using Proteome Discoverer (2.5) to interpret and quantify the relative amounts in a label free quantification manner. Data was searched against the Homo Sapiens proteome downloaded from Uniprot on 7/30/2020. Trypsin was set as the protease with up to two missed cleavages allowed. Carbamidomethylation of cysteine residues was set as a fixed modification. Oxidation of methionine and protein N-terminal acetylation were set as variable modifications. A precursor mass tolerance of 10 ppm and a fragment ion quantification tolerance of 0.05 Da were used. Data was quantified using the minora feature detector node within Proteome Discoverer.