Computational protocol: A Novel Role for Pro-Coagulant Microvesicles in the Early Host Defense against Streptococcus pyogenes

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Protocol publication

[…] Protein digestion was carried out as previously described . The resulting peptide mixtures were concentrated using spin-columns from Harvard Apparatus using the manufactures' instructions.The hybrid Orbitrap-LTQ XL mass spectrometer (Thermo Electron, Bremen, Germany) was coupled online to a split-less Eksigent 2D NanoLC system (Eksigent technologies, Dublin, CA, USA). Peptides were loaded with a constant flow rate of 10 µl/min onto a pre-column (Zorbax 300SB-C18 5×0.3 mm, 5 µm, Agilent technologies, Wilmington, DE, USA) and subsequently separated on a RP-LC analytical column (Zorbax 300SB-C18 150 mm×75 µm, 3.5 µm, Agilent technologies) with a flow rate of 350 nl/min. The peptides were eluted with a linear gradient from 95% solvent A (0.1% formic acid in water) and 5% solvent B (0.1% formic acid in acetonitrile) to 40% solvent B over 55 minutes. The mass spectrometer was operated in the data-dependent mode to automatically switch between Orbitrap-MS (from m/z 400 to 2000) and LTQ-MS/MS acquisition. Four MS/MS spectra were acquired in the linear ion trap per each FT-MS scan which was acquired at 60,000 FWHM nominal resolution settings using the lock mass option (m/z 445.120025) for internal calibration. The dynamic exclusion list was restricted to 500 entries using a repeat count of two with a repeat duration of 20 seconds and with a maximum retention period of 120 seconds. Precursor ion charge state screening was enabled to select for ions with at least two charges and rejecting ions with undetermined charge state. The normalized collision energy was set to 30%, and one microscan was acquired for each spectrum.The data analysis was performed as previously described . Briefly, the MS2 spectra were searched through the X! Tandem 2008-05-26 search engine against the human protein database. The search was performed with semi-tryptic cleavage, specificity, 1 missed cleavages, mass tolerance of 25 ppm for the precursor ions and 0.5 Da for fragment ions, methionine oxidation as variable modification and cysteine carbamidomethylation as fixed modification. The database search results were further processed using the Trans-Proteomic pipeline, version 4.4.0 . […]

Pipeline specifications

Software tools X! Tandem, TPP
Application MS-based untargeted proteomics
Organisms Mus musculus, Streptococcus pyogenes
Diseases Communicable Diseases, Infection
Chemicals Fibrinogen