Computational protocol: A proteomic approach to the development of DIVA ELISA distinguishing pigs infected with Salmonella Typhimurium and pigs vaccinated with a Salmonella Typhimurium-based inactivated vaccine

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Protocol publication

[…] 100 μg of proteins (measured with Pierce™ BCA Protein Assay Kit, Thermo Scientific, USA) was used for each sample preparation with the FASP (filter-aided sample preparation) method []. Each sample was washed five times with 8 M urea (Serva, Germany) in Vivacon 500 centrifugal tubes (Sartorius Stedim, Germany) with 10,000 MWCO membrane filter. Dithiothreitol (10 mM, Sigma-Aldrich, USA) and iodoacetamide (50 mM, Serva, Germany) in triethylammonium bicarbonate buffer (25 mM, Sigma-Aldrich, USA) were used for reduction and alkylation, respectively. Proteins were then digested with trypsin (Promega, USA) in 1:50 ratio, for one hour, at 37 °C and then overnight at 25 °C. After centrifugation, the eluate with digested peptides was evaporated (DNA120 SpeedVac, Thermo Savant, USA), peptide pellet was resuspended in 0.1 % aqueous formic acid (Sigma-Aldrich, USA) which serves as a mobile phase for liquid chromatography (UltiMate 3000 RSLCnano, Dionex -Thermo Scientific, USA). For separation and elution of peptides, 2-h gradient with increasing (0 min - 4 %, 4 min - 4 %, 98 min - 45 %, 98.5 min - 90 %, 112 min - 90 %, 112.5 min - 4 %, 120 min - 4 %) concentrations of acetonitrile (0.1 % formic acid in acetonitrile, Sigma-Aldrich, USA) at a flow rate of 300 nl/min was used. Peptides were separated on a 25 cm column (Acclaim PepMap RSLC C18, 2 μm, 100 Å, 75 μm I.D., Thermo Scientific, USA). uHPLC was connected to EASY-Spray ion source and Orbitrap Velos Pro mass spectrometer (Thermo Scientific, USA). A survey scan over the m/z range 390–1700 was used to identify protonated peptides with charge states of at least 2, which were automatically selected for data-dependent MS/MS analysis and fragmented by collision with helium gas. Ten fragment mass spectra after each full scan were recorded. Measured spectra were then searched using Proteome Discoverer (version 1.4, Thermo Scientific, USA) software with Mascot (Matrix Science, USA) or Sequest HT (Thermo Scientific, USA) as a searching algorithm. Oxidation of methionine as a dynamic, and carbamidomethylation of cysteine as a static modification was used. Precursor and fragment mass tolerances were set up as 10 ppm and 0.5 Da, respectively. Swiss-Prot database for Salmonella strain was used (based on 2014/06 release) in Mascot and Uniprot database for Salmonella strain (from 2013/04) was used in Sequest HT. Only peptides with a false discovery rate less than 0.05 were considered as well identified. Proteins that were uniquely found in each sample - proteins bound to the IgG fraction from sera from either vaccinated or infected animals, but not from control pigs were then pinpointed as candidate antigens for serological discrimination. […]

Pipeline specifications

Software tools Proteome Discoverer, Comet
Application MS-based untargeted proteomics
Organisms Salmonella enterica subsp. enterica serovar Typhimurium, Sus scrofa
Diseases Infection, Salmonella Infections