Computational protocol: Exosomes as nanocarriers for systemic delivery of the Helicobacter pylori virulence factor CagA

Similar protocols

Protocol publication

[…] Exosomal proteins were lysed in Laemmli sample buffer and reduced with 10 mM Tris (2-carboxyethyl) phosphine (Sigma-Aldrich) for 30 min at 37 °C, followed by alkylation with 50 mM iodoacetamide (Sigma-Aldrich) for 45 min in the dark at 25 °C. Proteins were separated on SDS-PAGE and stained with CBB. After desalting and drying excised gel bands, proteins were digested with Trypsin GOLD (Promega, Madison, WI, USA) at 37 °C for 16 h. The resulting peptides were extracted and analysed using an LTQ-Orbitrap-Velos mass spectrometer (Thermo Fisher Scientific) combined with an UltiMate 3000 RSLC nano-flow HPLC system (DIONEX Corporation). Protein identification analysis was performed using a SEQUEST database search with Proteome Discoverer 1.4 software (Thermo Fischer Scientific). The tandem mass spectrometry (MS/MS) spectra were searched against the Homo sapiens + Helicobacter pylori protein database in SwissProt. A false discovery rate of 1% and peptide rank of 1 were set for peptide identification filters. Extracted ion chromatograms were depicted with mass tolerance at 1 ppm. […]

Pipeline specifications

Software tools Comet, Proteome Discoverer
Application MS-based untargeted proteomics
Organisms Helicobacter pylori, Homo sapiens
Diseases Gastrointestinal Diseases, Infection, Genetic Diseases, Inborn