Computational protocol: Brachyspira hyodysenteriae and B. pilosicoli Proteins Recognized by Sera of Challenged Pigs

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Protocol publication

[…] Silver stained bands corresponding to the reactive bands detected in the immunoblots were excised and digested using an automatic device (DigestPro MS, Intavis, Cologne, Germany). The process involved reduction with dithiothreitol, derivatization with iodoacetamide, and enzymatic digestion with trypsin (37°C, 8 h) (Casanovas et al., ). The tryptic digests were evaporated and redissolved in 5 μl of methanol/water/trifluoroacetic acid (30/70/0.1 v/v).Proteins in the tryptic digests (0.5 μl) were identified by MALDI-TOF peptide mass fingerprinting combined with MS/MS ion search in a 4800 TOF/TOF mass spectrometer (ABSciex, Barcelona, Spain) in the reflectron mode. The spectra were externally mass calibrated using a standard peptide mixture. Alpha-cyano-4-hydroxycinnamic acid (3 mg/ml) was used as the matrix. The five signals with the greatest intensity in each MALDI-TOF spectrum were automatically analyzed by TOF/TOF. The combined TOF and TOF/TOF spectra were interpreted by database search (Mascot, Matrix Science, MA, USA) using the following parameters: peptide mass tolerance, 50 ppm; fragment mass tolerance, 0.5 Da; fixed modification, carbamidomethyl cysteine; variable modification, oxidation of methionine; significance threshold of the MOWSE score, p < 0.05. All identifications were manually validated.Samples which did not produce a positive identification by MALDI were reanalysed by LC-MS/MS in a Velos-LTQ or an Orbitrap-XL mass spectrometer (Thermo Fisher Scientific) equipped with a microESI ion source. Four microliters of each sample digest were diluted to 20 μl with 5% methanol and 1% formic acid, and loaded into a chromatographic system consisting of a C18 preconcentration cartridge (Agilent Technologies) connected to a 15-cm long, 150 μm i.d. (Velos-LTQ) or 100 μm i.d. (Orbitrap-XL) C18 column (Nikkyo Technos Co.). The separation was performed at 1 μl/min (Velos-LTQ) or 0.4 μl/min (Orbitrap XL) in a 30-min gradient from 3 to 40% acetonitrile (solvent A: 0.1% formic acid, solvent B: acetonitrile 0.1% formic acid). The instruments were operated in the positive ion mode with a spray voltage of 1.5 kV. The spectrometric analysis was performed in a data dependent mode. The scan range for full scans was m/z 400–1,800. The LC-MS/MS spectra were searched using SEQUEST (Proteome Discoverer v1.4, Thermo–Fisher Scientific) with the following parameters: peptide mass tolerance, 1 Da (Velos-LTQ) or 20 ppm (Orbitrap-XL); fragment tolerance, 0.6 Da; enzyme, trypsin; two missed cleavages allowed; dynamic modification, methionine oxidation (+16 Da); fixed modification, cysteine carbamidomethylation (+57 Da). The peptide identifications were filtered at 0.1% FDR and only proteins identified with two or more peptides and peptide rank 1 were considered. Relative abundance of the identified proteins in each sample was roughly estimated from the product of the total peptide sequence matches pointing to that protein and its sequence coverage. The group of more abundant proteins bearing more than 80% of the total abundance in the sample were considered for discussion (Full data is available in Supplementary Tables –).Searches for the MALDI and LC-MS/MS methods described above were carried out against the Uniprot database (2015_11 version) restricted to Brachyspira. When results pointed to indistinguishable different accessions to the same protein in different strains, the accession for the reference ATCC strains was reported in Table , Supplementary Tables , . […]

Pipeline specifications

Software tools Comet, Proteome Discoverer
Application MS-based untargeted proteomics
Organisms Brachyspira hyodysenteriae, Brachyspira pilosicoli, Sus scrofa
Diseases Dysentery