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Protocol publication

[…] After immunoprecipitation, proteins were separated on SDS–PAGE gels (Invitrogen) and stained with colloidal blue staining (LabSafe GEL BlueTM GBiosciences). Gel slices were excised and proteins were reduced with 10 mM DTT prior to alkylation with 55 mM iodoacetamide. After washing and shrinking the gel pieces with 100% MeCN, in-gel digestion was performed using trypsin (Promega) overnight in 25 mM NH4HCO3 at 30°C.Peptides were extracted and analyzed by nano-LC-MS/MS using an Ultimate 3000 system (Dionex S.A.) coupled to an Orbitrap Fusion mass spectrometer (Q-OT-qIT, Thermo Fisher Scientific). Samples were loaded on a C18 precolumn (300 µm inner diameter × 5 mm; Dionex) at 20 µl/min in 5% MeCN, 0.1% TFA. After a desalting for 3 min, the precolumn was switched on the C18 column (75 μm i.d. × 50 cm, packed with C18 PepMap™, 3 μm, 100 Å; LC Packings) equilibrated in solvent A (2% MeCN, 0.1% HCO2H). Bound peptides were eluted using a 150 min linear gradient (from 5 to 30% (v/v)) of solvent B (80% MeCN, 0.085% HCO2H) at a 150 nl/min flow rate and an oven temperature of 40°C. We acquired Survey MS scans in the Orbitrap on the 400–1500 m/z range with the resolution set to a value of 240,000 and a 4 × 105 ion count target. Each scan was recalibrated in real time by co-injecting an internal standard from ambient air into the C-trap. Tandem MS was performed by isolation at 1.6 Th with the quadrupole, HCD fragmentation with normalized collision energy of 35, and rapid scan MS analysis in the ion trap. The MS2 ion count target was set to 104 and the max injection time was 200 ms. Only those precursors with charge state 2–7 were sampled for MS2. The dynamic exclusion duration was set to 60 s with a 10 ppm tolerance around the selected precursor and its isotopes. The instrument was run in top speed mode with 3 s cycles.Data were acquired using the Xcalibur software (v 3.0.63) and the resulting spectra were interrogated by the MascotTM Software through Proteome Discoverer (v 1.4.0.288, Thermo Scientific) with the SwissProt Mus musculus database (20140402, 16,671 sequences). We set carbamidomethyle cysteine, oxidation of methionine and N-terminal acetylation as variable modifications. We set specificity of trypsin digestion and allowed 2 missed cleavage sites and we set the mass tolerances in MS and MS/MS to 2 ppm and 0.5 Da, respectively. The resulting Mascot files were further processed by using myProMS (v 3.0) and the estimated false discovery rate (FDR) by automatically filtering the Mascot score of all peptide identifications was less than 0.5%. […]

Pipeline specifications

Software tools Proteome Discoverer, myProMS
Application MS-based untargeted proteomics
Organisms Mus musculus