Computational protocol: A gel-free proteomic-based method for the characterization of Bordetella pertussis clinical isolates

Similar protocols

Protocol publication

[…] Protein identification was achieved by using nanoflow liquid chromatography (nano-LC), data-dependent tandem mass spectrometry, and database searching. A pulled-needle, fused silica capillary (365 µm O.D. by 75 µm I.D.) (New Objective, Inc., Woburn, MA) was packed with 10 cm of 5 µm Symmetry 300 reverse-phase packing material (Waters Inc., Bedford, MA). Protein digests were loaded onto the analytical column and separated by gradient elution using an Eksigent 2D nanoLC system (Eksigent Technologies, Inc, Dublin, CA). The mobile phase solvents consisted of (solvent A) 0.2% formic acid (Thermo Scientific, Rockford, IL), 0.005% trifluoroacetic acid (Sigma-Aldrich) in water (Burdick and Jackson, Muskegon, MI), and (solvent B) 0.2% formic acid, 0.005% trifluoroacetic acid in acetonitrile (Burdick and Jackson). The gradient flow was set at 400 nl/min. The profile consisted of a hold at 5% B for 5 min followed by a ramp to 30% B over 100 min, then a ramp up to 90% B in 5 min and a hold at 90% for 2 min before returning to 5% B in 2 min and re-equilibration at 5% B for 20 min. After chromatography, peptides were introduced into an LTQ Orbitrap tandem mass spectrometer (Thermo Scientific, San Jose, CA). A 2.0 kV voltage was applied to the nano-LC column. The mass spectrometer was programmed to perform data-dependent acquisition by scanning the mass range from mass-to-charge (m/z) 400 to 1600 at a nominal resolution setting of 60,000 for parent ion acquisition in the Orbitrap. Most tryptic peptides fall within the stated m/z range and served as the basis for this selection. For MS/MS analysis the mass spectrometer chose the top 10 most intense ions with two or more charges. Singly charged ions were rejected for MS/MS as these ions are likely due to detergents or other sample additives. In particular for a data-dependent acquisition, time is better utilized acquiring for doubly and triply charged amino acids, which predominantly have greater sequence specificity since they are larger peptides and thus provide a higher likelihood in which to uniquely identify a protein.All tandem mass spectra were extracted from the raw data file using Mascot Distiller (Matrix Science, London, UK; version 2.2.1.0) and searched using Mascot (version 2.2.0). Mascot was set up to search using the entire NCBInr database or a modified NCBInr database created to search “Bordetella”- or “pertussis”- recognized proteins in which trypsin is used as the digestion agent. Mascot was searched with two missed cleavages, a fragment ion tolerance mass of 0.80 Da, and a parent ion tolerance of 200 ppm, while oxidation was selected as a variable modification. Scaffold (Proteome Software, Portland, OR) was used to validate MS/MS based peptide and protein identifications.Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm (). Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least two identified peptides (). With these stringent parameters of Peptide Prophet and Protein Prophet within the Scaffold software, the probability of a wrong assignment is below 0.1%. PSORTb subcellular scores were used to predict and localize identified EMF proteins (http://www.psort.org/psortb/) (). Lastly, KEGG identifiers using NCBI Gi accession numbers were employed to assign functions to each of the identified proteins http://www.genome.jp/kegg/kegg3.html (). […]

Pipeline specifications

Software tools Mascot Distiller, PSORTb
Applications MS-based untargeted proteomics, Protein sequence analysis
Organisms Bordetella pertussis, Homo sapiens
Diseases Respiratory Insufficiency, Whooping Cough