Identification of Salmonella enterica serovar Typhimurium UK-1 antigens in sonicated bacteria
Proteomic analysis of Salmonella enterica serovar Typhimurium UK-1 proteins in lysed bacteria preparation.
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Stationary Salmonella enterica serovar Typhimurium UK-1 culture was spun at 13,000 x g at 4ºC, and the obtained pellet was washed with PBS containing 5 mM EDTA, spun down again, and then washed with PBS alone. The cells were sonicated by using the Sonifier Cell Disruptor (Heat Systems-Ultrasonics Inc., Plainview, NY), followed by pelleting the debris 13,000 x g at 4ºC to obtain protein-containing supernatant. Filter sterilization of such obtained antigen (Ag) preparation was carried out by filtering through a 0.22-µm PES filter. The antigen preparations were incubated on LB agar plates to verify that no live cells remain. Protein concentration in antigens was determined by BCA assay. 25 µg per sample was used, and three replicates per sample type were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The entire lane was excised by scalpel and digested by using in-gel trypsin digestion. The peptide samples were analyzed by 250-mm Ultrahigh-Performance Liquid Chromatography coupled to Orbitrap Fusion mass spectrometer (Thermo Scientific). The liquid chromatography was done using the Thermo EASY nano-LC system, where 20-mm C16 pre-column (Thermo Scientific) was used to rid of impurities, after which the samples were separated by using a reversed-phase C18 analytical column with the 100Å pore (Thermo Scientific, Acclaim PepMap 100 C18 LC Column). The LC system was interfaced directly with Orbitrap Fusion mass spectrometer (Thermo Scientific). MS data were acquired at 120K resolution by Orbitrap detector. Ions were isolated by a quadrupole, prioritizing the most intense ions and injecting ions for all available parallelizable time. Fragmentation was done by using collision-induced dissociation (CID), at a collision energy of 35% and activation time of 10 ms, where the AGC target of 10000. The MS/MS data were detected by the ion trap. Tandem MS spectra were extracted, charge state deconvoluted, and deisotoped by Proteome Discoverer (Thermo Scientific). MS/MS samples were analyzed using Sequest and X! Tandem. Sequest was set up to search FASTA Salmonella Typhimurium Uniprot database containing a common list of contaminants database assuming the digestion enzyme trypsin. Sequest and X! Tandem were both searched with a fragment ion mass tolerance of 1.00 Da and a parent ion tolerance of 10.0 ppm. Carbamidomethyl of cysteine was specified as a fixed modification. Deamidation of asparagine, oxidation of methionine and acetyl of the N-terminus were specified as variable modifications. The Scaffold software was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if established at greater than 95.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at >95.0% probability and minimum two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm and protein grouping was done.