Computational protocol: ATP-mediated Events in Peritubular Cells Contribute to Sterile Testicular Inflammation

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Protocol publication

[…] Cell lysis was performed with an ultrasonic device (10,000 kJ, Sonoplus GM3200 with BR30 cup booster, Bandelin, Berlin, Germany) in 7 µl 8 M urea/0.4 M NH4HCO3 per 100,000 cells. Samples were further centrifuged through QIA-Shredder devices (Qiagen, Hilden, Germany). Protein concentrations were determined by a Bradford assay and the samples were adjusted with 8 M urea/0.4 M NH4HCO3 to a concentration of 2 mg/ml protein. Cysteine residues were reduced for 30 min using DTE at a concentration of 4.5 mM and blocked with iodoacetamide (final concentration 10 mM) for 30 min in the dark. Samples were diluted with water to a concentration of 1 M urea and trypsinised overnight at 37 °C using 20 ng porcine trypsin (Promega, Madison, WI, USA) per µg of protein. For LC-MS/MS analysis an Ultimate 3000 chromatography system (Thermo Scientific, Waltham, MA, USA) coupled to a TripleTOF 5600+ mass spectrometer (Sciex, Concord, Canada) was used. 2.5 µg of peptides diluted in 0.1% formic acid (FA) were injected on a trap column (Acclaim PepMap 100, μ-Precolumns, 5 mm × 300 μm, 5 μm particles, Thermo Scientific) and separated at a flow rate of 200 nL/min (Acclaim PepMap RSLC C18, 75 μm × 50 cm, 2 μm; Thermo Scientific). The LC method consisted of consecutive gradients from 5% to 25% solvent B (0.1% FA, 100% ACN) in 290 min and from 25% to 50% solvent B in 30 min. MS spectra were acquired using a top 70 method (mass range m/z 400–1250, rolling collision energy activated). Peptide identification and LFQ quantification was performed with MaxQuant V1.5.1 using the Homo sapiens subset of the UniProt database. For identification, the MaxQuant default parameters for Sciex TOF instruments were used and the FDR at the peptide and protein level was set to 1%. Student’s t-test was performed with the Perseus module of MaxQuant. To handle missing values the Perseus imputation feature was used in cases where proteins were detected in at least three replicates of one group. Enrichment analysis was performed with the STRING analysis tool (http://string-db.org, version 10.0) using default options. […]

Pipeline specifications

Software tools MaxQuant, Perseus
Application MS-based untargeted proteomics