Computational protocol: The Susceptibilities of Respiratory Syncytial Virus to Nucleolin Receptor Blocking and Antibody Neutralization Are Dependent upon the Method of Virus Purification

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Protocol publication

[…] All samples were reduced, denatured, and run into a Biorad TGX precast SDS-PAGE gel. The Coomassie-stained bands were then excised and in-gel trypsin digestion was performed. Briefly, the excised gel bands were destained twice in 100 mM ammonium bicarbonate/acetonitrile (50:50). Samples were reduced (10 mM β-mercaptoethanol in 100 mM bicarbonate) and alkylated (55 mM iodoacetamide in 100 mM bicarbonate). After dehydration, enough trypsin (6 ng/µL) was added to cover the gel pieces and the digestion proceeded overnight at room temperature. Tryptic peptides were first extracted from the gel using 97% water/2% acetonitrile/1% formic acid followed by a second extraction using 50% of the first extraction buffer and 50% acetonitrile. Fractions containing tryptic peptides dissolved in aqueous 25% v/v ACN and 1% v/v formic acid were resolved and ionized by using nanoflow HPLC (Easy-nLC II, Thermo Scientific) coupled to the LTQ XL-Orbitrap hybrid mass spectrometer (Thermo Scientific). Nanoflow chromatography and electrospray ionization were accomplished by using a New Objective PicoFrit fused silica capillary column (ProteoPepII, C18) with 100 μm inner diameter (300 Å, 5 μm). Peptide mixtures were injected onto the column at a flow rate of 3000 nL/min and resolved at 500 nL/min using 35 min linear gradients from 0 to 45% v/v aqueous ACN in 0.2% v/v formic acid. The mass spectrometer was operated in data-dependent acquisition mode, recording high-accuracy and high-resolution survey Orbitrap spectra using external mass calibration, with a resolution of 30,000 and an m/z range of 400–2000. The fourteen most intense multiply charged ions were sequentially fragmented by using collision-induced dissociation, and spectra of their fragments were recorded in the linear ion trap; after two fragmentations, all precursors selected for dissociation were dynamically excluded for 60 s. Data was processed using Proteome Discoverer 1.4 (Thermo Scientific) and a Uniprot human database was searched using SEQUEST (Thermo Scientific). The search parameters included a precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.8 Da. Peptides were searched with carbamidomethyl cysteine as a static modification and oxidized methionine, deamidated glutamine, and asparagine as dynamic modifications. […]

Pipeline specifications

Software tools Proteome Discoverer, Comet
Application MS-based untargeted proteomics
Organisms Respiratory syncytial virus, Human poliovirus 1 Mahoney, Rous sarcoma virus
Chemicals Sucrose, Superoxides