Computational protocol: Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA

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Protocol publication

[…] Two biological replicates were prepared for MS analysis. 20 µL of each sample was digested following extraction of the proteins. Tryptic peptides were desalted using PierceTM C18 spin columns (Thermo Fisher Scientific) and analyzed using a Dionex Ultimate 3000 Nano LC System (Sunnyvale, CA) coupled to a Bruker Maxis II Q-TOF mass spectrometer (Bremen, Germany) with CaptiveSpray nanoBooster ionization source. In case of the first vesicle isolation separation of the peptides was achieved online using a 15 cm Acclaim Pepmap RSLC nano HPLC column (Thermo Fisher Scientific) following trapping on an Acclaim™ PepMap100™ C18 Nano-Trap column (5 µm, 100 Å, 100 µm × 20 mm, Thermo Fisher Scientific). Peptides originating from the second vesicle isolation were separated online using a 25 cm Waters Peptide BEH C18 nanoACQUITY 1.7 µm particle size UPLC column following the same trapping conditions. Data were processed with ProteinScape 3.0 software (Bruker Daltonik GmbH, Bremen, Germany). Protein identification was performed against Swissprot database (2015_08) using Mascot (Matrix Science, London, UK; version Mascot 2.5) and X! Tandem (The GPM, thegpm.org; version 2007.01.01.1) search engines. The following parameters were applied: Homo sapiens taxonomy, trypsin enzyme, 7 ppm peptide mass tolerance, 0.05 Da fragment mass tolerance, 2 missed cleavages. Carbamidomethylation was set as fixed modification, while deamidation (NQ), oxidation (M) and pyro-carbamidomethylation (N-term C) as variable modifications. Scaffold (version Scaffold_3_00_07, Proteome Software Inc., Portland, OR) software was used to validate peptide and protein identifications as previously published. Label-free quantification of histones was performed using MaxQuant software version 1.5.3.30. […]

Pipeline specifications

Software tools X! Tandem, MaxQuant
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Bacterial Infections, Mycoplasma Infections
Chemicals Ciprofloxacin, Fluoroquinolones