Computational protocol: HIV-1 and Human PEG10 Frameshift Elements Are Functionally Distinct and Distinguished by Novel Small Molecule Modulators

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Protocol publication

[…] Peptides containing the site of frameshifting were identified by nanoflow uHPLC-coupled tandem mass spectrometry as described previously []. In brief protein bands containing either the PEG10 or HIV-1 frameshift products were excised and in-gel digested with trypsin []. Tryptic peptides were then separated by nanoflow RP-uHPLC on an in-house packed emitter tip column (75 μm ID silica tubing packed with 3 μm C18 beads at a length of 12 cm) in line coupled to the nanospray source of a LTQ Orbitrap XL mass spectrometer (Thermo Scientific, San Jose, CA) using a gradient developed from 5% solvent B (0.2% formic acid in acetonitrile) in solvent A (0.2% formic acid in water) to 30% solvent B in solvent A over 12 min, followed by an increase to 95% solvent B in solvent A over 6 min. The LTQ Orbitrap mass spectrometer was operated in data dependent mode to allow for the acquisition of one full MS spectrum in the Orbitrap analyzer at a resolution of 60,000 (at m/z 400) and 5 ion trap CID fragment ion spectra per cycle. Raw data was processed through the Proteome Discoverer software using default settings. Spectra were then searched against a combined amino acid sequence database containing all SwissProt/UniProt sequence entries (546,057 entries) and 8 custom-made sequences covering four predicted sites of frameshifting for HIV-1 and PEG10 using the Mascot (http://www.matrixscience.com/server.html) and SequestHT (Thermo Scientific, San Jose, CA) search engines. The searches were set up for tryptic peptides including the variable modifications of oxidized methionine, carboxyamidomethyl cysteine and deamidated asparagine and glutamine. For both search engines the Percolator algorithm [] was used to adjust the score threshold for significant peptide identification at a false discovery rate of <1%. […]

Pipeline specifications

Software tools Proteome Discoverer, Percolator
Application MS-based untargeted proteomics
Organisms Human immunodeficiency virus 1, Homo sapiens
Diseases HIV Infections