Computational protocol: Mucosal fluid glycoprotein DMBT1 suppresses twitching motility and virulence of the opportunistic pathogen Pseudomonas aeruginosa

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Protocol publication

[…] Mass spectrometry (MS) was performed at the Proteomics/Mass Spectrometry Laboratory, University of California, Berkeley. A nano LC column was packed in a 100 μm inner diameter glass capillary with an emitter tip. The column consisted of 10 cm of Polaris C18 5 μm packing material (Varian, Agilent, CA), followed by 4 cm of Partisphere 5 SCX (Whatman, Sigma-Aldrich, MO). The column was loaded by use of a pressure bomb and washed extensively with buffer A (see below). The column was then directly coupled to an electrospray ionization source mounted on a Thermo-Fisher LTQ XL linear ion trap mass spectrometer. An Agilent 1200 HPLC equipped with a split line to deliver a flow rate of 300 nl/min was used for chromatography. Peptides were eluted using a 4-step MudPIT procedure []. Buffer A was 5% acetonitrile/ 0.02% heptaflurobutyric acid (HFBA); buffer B was 80% acetonitrile/ 0.02% HFBA. Buffer C was 250 mM ammonium acetate/ 5% acetonitrile/ 0.02% HFBA; buffer D was same as buffer C, but with 500 mM ammonium acetate.Protein identification and quantification were done with Integrated Proteomics Pipeline (IP2, Integrated Proteomics Applications, Inc. San Diego, CA) using ProLuCID/Sequest, DTASelect2 and Census [–]. Tandem mass spectra were extracted into ms1 and ms2 files from raw files using RawExtractor [], and searched against the human protein database plus sequences of common contaminants, concatenated to a decoy database in which the sequence for each entry in the original database was reversed []. […]

Pipeline specifications

Software tools ProLuCID, Comet, DTASelect
Application MS-based untargeted proteomics
Organisms Pseudomonas aeruginosa, Homo sapiens, Mus musculus
Diseases Infection, Opportunistic Infections
Chemicals Cyclic AMP