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
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.