Computational protocol: Intrinsically-disordered N-termini in human parechovirus 1 capsid proteins bind encapsidated RNA

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Protocol publication

[…] The RCAP assay was done as described previously, using two moles of recombinant protein to one mole of RNA. Formaldehyde was then added to a final concentration of 0.1% and incubated for 10 min at room temperature. Glycine (0.2 M) was added to quench the formaldehyde and incubated for 10 min at room temperature, then crosslinked protein-RNA complexes were subjected to trypsin digestion using sequencing-grade trypsin (Trypsin Gold, Promega) for 16 hours using a 1:20 w/w ratio of enzyme to capsid. RNA-peptide complexes were then selectively precipitated using lithium chloride, and to facilitate mass spectrometric analysis peptide-RNA conjugates were reversed by supplementing the buffer with 150 mM sodium chloride and heating at 65 οC for 1 h. Control reactions lacking formaldehyde, and when using the recombinant protein lacking RNA, were performed in parallel.RCAP assays performed with chemically synthesized peptides with an N-terminal carboxyfluorescein (Zhejiang, China). Each peptide was purified using HPLC to at least 95% purity and the molecular mass was confirmed by mass spectrometry to be accurate to within 1 Dalton. The peptides were present at two molar excess to the HPeV1 RNA and crosslinked as described above. To facilitate ionization and proteolysis, cysteine-containing peptides were supplemented with 200 mM Tris-HCl (pH 9.0), 5 mM TCEP, and alkylated with 20 mM bromoethylamine prior to digestion as described previously,. The crosslinked peptide-RNAs were digested with either 1 μg trypsin, chymotrypsin, or Asp-N as specified (Pierce MS-grade protease) overnight at 37 °C. Controls included RNAs and peptides without formaldehyde, and proteolyzed samples.For the virion RCAP data, peptides were analyzed using an LTQ Velos Pro dual-pressure linear ion trap mass spectrometer (ThermoFisher Scientific). Tryptic digests were injected into a C18 column (Dionex UltiMate 3000 HPLC) and eluted using a linear gradient of 2–45% acetonitrile in water with 0.1% formic acid, over a 90 minute gradient. The flow rate was 50 uL/min, and effluent was electro-sprayed into the LTQ. Tandem mass spectra were obtained using collision-induced dissociation. Experiments using recombinant protein were analyzed on an LTQ Orbitrap mass spectrometer (ThermoFisher scientific), and separated similarly using an Accela HPLC. Peptide RCAP data was generated on a Bruker Autoflex III MALDI-ToF and analyzed using GPMAW (General Protein/Mass Analysis for Windows, Supplementary Figure ).Database searches used searchGUI and the peptide sequences were compiled with peptideshaker,. Databases were created using HPeV1 protein sequences appended to a FASTA file containing the uniprot human proteome and the common repository of adventitious proteins database. Linear ion trap data was searched using a 0.3 Da mass error, and Orbitrap using 10 ppm error. Peptides appearing in the negative controls were used to gauge the quality of LiCl precipitation, and results were discounted if peptides were detectable. Only identifications with a confidence score of 98% or better were included. The identified peptides for the virion RCAP are listed in Supplementary Table  and the top 12 hits for each protein were considered as most significant. This accounted for 81% of the unique observations. Disorder predictions were retrieved from the PONDR server (http://www.pondr.com/) using the VL-XT algorithm. […]

Pipeline specifications

Software tools GPMAW, SearchGUI, PeptideShaker
Databases cRAP
Application MS-based untargeted proteomics
Organisms Homo sapiens, Hot pepper alphaendornavirus, Human poliovirus 1 Mahoney