Computational protocol: Protein Composition of the Bovine Herpesvirus 1.1 Virion

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Protocol publication

[…] Spectral data were collected using an Orbitrap LTQ Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) linked with an UltiMate 3000 nano flow HPLC system (Thermo Fisher Scientific). A total of two µg of the trypsin-digested protein was loaded on a reversed phase fused silica Acclaim PepMap C18 column, measuring 75 µm × 150 mm (Thermo Fisher Scientific). Peptides were separated and eluted using a constant flow rate of 0.3 microliters per minute in a 60-min long linear gradient of acetonitrile (in 0.1% formic acid): 2%–55% for 35 min, 95% for 10 min, 2% for 15 min. Peptides were detected by a linear trap mass detector, in the data-dependent acquisition mode with dynamic exclusion being applied. Eight scan events were employed: one MS scan (m/z range: 300–2000) followed by seven tandem mass spectrometry (MS/MS) scans for the seven most intense ions detected in the MS scan. Selected parameters were set as follows: Normalized collision energy: 35%; automatic gain control “on” with MSn Target 4 × 104; isolation width (m/z): 1.5; capillary temperature 170 °C; spray voltage 1.97 kV. [].The .raw files were searched using the SEQUEST HT algorithm of the Proteome Discoverer 2.1 SP1 software (Thermo Fisher Scientific) with given parameters: Lowest and highest charge: +1 and +3, respectively; minimum and maximum precursor mass: 300 and 6000 Da, respectively; minimum S/N ratio: 3; enzyme: trypsin; maximum missed cleavages: 2; dynamic modifications: cysteine carbamidomethylation (+57.021), methionine oxidation (+15.995) and methionine dioxidation (+31.990).The spectral data were matched against the target BoHV type 1 protein database downloaded from NCBI (National Center for Biotechnology Information) (www.ncbi.nlm.nih.gov), and Bos taurus referenced protein database downloaded from UniProt (Universal Protein Resource) (www.uniprot.org), both as of November 2015. To calculate the false discovery rate (FDR), the software uses the decoy database created by reversing all protein sequences of the target database. Matches were filtered by value of FDR <1.0%. One “hit” identifications were excluded, however proteins identified by at least one unique peptide were accepted, if the peptide was detected multiple times across the replicates. The exponentially modified protein abundance indexes (emPAI), calculated automatically by Proteome Discoverer, were used to approximate the absolute amounts of identified proteins within the sample.The inclusion/exclusion criterion for host proteins in virion preparations is as follows: host proteins had to appear in at least two of the three samples to be included. At the same time, proteins detected in any of the uninfected supernatants were automatically rejected from the list. […]

Pipeline specifications

Software tools Comet, Proteome Discoverer
Databases UniProt
Application MS-based untargeted proteomics
Organisms Bos taurus, Bovine alphaherpesvirus 1
Diseases Respiratory Insufficiency