Computational protocol: A quantitative proteomic screen of the Campylobacter jejuni flagellar dependent secretome

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Protocol publication

[…] The unlabelled, or SILAC labelled samples were reduced with tris(2-carboxyethyl) phosphine (TCEP) then alkylated with iodoacetamide (Sigma) followed by digestion by trypsin (Thermo Fisher Scientific) overnight at 37 °C. 0.5 μg (unlabelled samples) or 1.5 μg (SILAC samples) of the digest were submitted for the nano LC-MS/MS analyses on an Ultimate 3000 RSLCnano System coupled to a LTQ Orbitrap Velos hybrid mass spectrometer equipped with a nanospray source. The peptides were first loaded and desalted on a PepMap C18 trap column (100 μm id × 20 mm, 5 μm) then separated on a PepMap C18 analytical column (75 μm id × 500 mm, 2 μm) over a 90 min (unlabelled samples) or 180 min (SILAC labelled samples) linear gradient of 4–32% CH3CN/0.1% formic acid (the HPLC, mass spectrometer and columns were all from Thermo Fisher Scientific). The Orbitrap mass spectrometer was operated in the standard “top 15 or top 10” data-dependant acquisition modes while the preview mode was disabled. The MS full scan was set at m/z 380–1600 with the resolution at 30,000 at m/z 400 and AGC at 1 × 106 with a maximum injection time at 200 msec. The 15, or 10, most abundant multiply-charged precursor ions, with a minimal signal above 3000 counts, were dynamically selected for CID fragmentation (MS/MS) in the ion trap, which had the AGC set at 5000 with the maximum injection time at 100 msec. The dynamic exclusion duration time was set for 60 s with ± 10 ppm exclusion mass width.The raw files were processed in MaxQuant (Version 1.5.2.8, www.MaxQuant.org) for both protein identification and protein quantification. The C. jejuni M1 protein database was a combination of those downloaded from UniprotKB (www.uniprot.org) of 11,168 (April 2015) and M1 (February 2015). Parameters used were mainly in default values with some modifications: trypsin with maximum 2 missed cleavage sites, peptide mass tolerance at first search was set at 20 ppm and main search was at 4.5 ppm, MS/MS fragment mass tolerance at 0.50 Da, and top 8 MS/MS peaks per 100 Da and a minimum peptide length of 7 amino acids were required. Fixed modification for Carbamidomethyl and variable modification for Acetyl (Protein N-term), Deamidated (NQ) and Oxidation (M) were used. False discovery rates (FDR) were estimated based on matches to reversed sequences in the concatenated target-decoy database. The maximum FDR at 1% was allowed for proteins and peptide spectrum matches (PSMs). Peptides were assigned to protein groups, a cluster of a leading protein(s) plus additional proteins matching to a subset of the same peptides. For protein quantification, the minimum ratio of two, from ‘unique and razor peptides’ was required, and Re-quantify was enabled but Match between runs was disabled. The protein FDR was set to 0.1%. The MaxQuant output was processed using Perseus (Version 1.5.2.6 www.MaxQuant.org). Protein groups that are only identified by site, or reverse matches and potential contaminants. Are filtered out. A log2 transformation of SILAC ratio was carried out, rows were filtered to contain a minimum of two values and filtered using a Benjamini-Hochberg FDR test to 0.05. The SILAC data is provided in , the label-free LC/MS data is provided in . […]

Pipeline specifications

Software tools MaxQuant, Perseus
Databases UniProtKB
Application MS-based untargeted proteomics
Organisms Campylobacter jejuni, Homo sapiens
Chemicals Amino Acids