Computational protocol: Assessing Protein Immunogenicity with a Dendritic Cell Line-Derived Endolysosomal Degradome

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Protocol publication

[…] Endosomes and lysosomes were isolated from DCs by differential centrifugation . Briefly, cells (107 cells/protein) were homogenized in 10 mmol L−1 Tris/acetate pH 7 containing 250 mmol L−1 sucrose using a Dounce tissue grinder (Sigma-Aldrich, St. Louis, MO) and centrifuged for 10 minutes at 6,000× g. To obtain a total microsomal fraction, postnuclear supernatants were ultracentrifuged (60 minutes at 100,000× g). Microsomal content was released by 5 freeze-thaw cycles on liquid nitrogen and room temperature respectively, and stored at −20°C. For microsome fingerprinting 100 ng of microsomal proteins were reduced, alkylated, and trypsinized using the Calbiochem® ProteoExtract® All-in-One Trypsin digestion kit (EMD, Gibbstown, NJ) before injection into a one-dimensional capillary HPLC system (Model U3000, Dionex Benelux, Amsterdam, The Netherlands), equipped with a low-pressure gradient micro-pump, a micro-autoinjector and a capillary PS-DVB monolithic separation column (150×0.2 mm id). A 300 min gradient of 0–40% ACN in 0.05% aqueous TFA was applied using a flow rate of 1 µl min−1. The chromatographic setup was coupled to an ESI-LTQ Orbitrap mass analyzer (Thermo Fisher Scientific GmbH, Bremen, Germany). Each sample was analyzed in triplicate, while the peptides identified in one run were excluded from data dependent decisions in the following runs by the use of exclusion lists. Spectra were generated in positive mode in a mass range of m/z 500–2000. Fragmentation of a maximum of three precursors was realized in the ion trap using collision-induced dissociation. The Mascot search engine (Matrix Science, London, UK) and the software Proteome Discoverer (Thermo Fisher Scientific GmbH, Bremen, Germany) were used for peptide identification with the following parameters: taxonomy, all entries; Variable modification, methionine oxidation; fixed modification, carbamidomethyl (C), Enzyme, trypsin; peptide tolerance, ±10 ppm; MS/MS tolerance, ± 0.3 Da; maximum missed cleavages, 1; Human and murine samples were searched against species-specific SwissProt databases. […]

Pipeline specifications

Software tools Mascot Server, Proteome Discoverer
Application MS-based untargeted proteomics
Organisms Mus musculus, Homo sapiens
Diseases Drug Hypersensitivity, Hypersensitivity