Shotgun proteomics (mouse)

Nuclear extracts of cells expressing Trnp1, FLAGTrnp1, delta1-16Trnp1 or FLAGdelta1-16Trnp1 were obtained by re-suspended cells for 30 min on ice in Tween20 lysis buffer (25 mM HEPES pH 8, 20 mM NaCl, 2 mM EDTA, 1 mM PMSF, 0.5% Tween20, 1X protease inhibitors, cOmplete, Roche), pelleted nuclei by centrifugation, discarding the cytoplasmic fraction (supernatant) and lysing nuclei by sonication with lysis buffer containing 500 mM NaCl. DNAseI (250U; SIGMA) was added to the nuclear pellet and incubated 1 hour at 37°C. Chromatin and cell debris were pelleted at 12000 rpm for 15min at 4°C and the supernatant containing the nuclear proteins were recovered in a low-protein binding Eppendorf. For co-immunoprecipitation, the nuclear protein extract was incubated end-over-end with 10 FLAG® M2 Magnetic Beads (SIGMA) at 4°C for 2 hours. Beads were then separated with a magnet and washed 4 times for 15 minutes at 4°C with washing buffer ((25 mM HEPES pH 7,6, 150 mM KCl, 12,5 mM MgCl2, 0,1 mM EDTA, 10% glycerol) + 0,1% NP-40). For Western blot analysis, proteins were eluted by cooking beads at 95°C with mixer at 600 rpm for 10 min in 2X Laemmli buffer and separated from the beads with a magnet. For LC-MS/MS beads were subsequently washed three times with 50mM NH4HCO3, incubated with 10 ng/µL trypsin in 1 M urea 50mM NH4HCO3 for 30 minutes, washed with 50mM NH4HCO3 again and digested ON in presence of 1mM DTT. Digested peptides were alkylated and desalted. For LC-MS/MS purposes, desalted peptides were injected in an Ultimate 3000 RSLCnano system (Thermo) and separated in a 15-cm analytical column (75μm ID with ReproSil-Pur C18-AQ 2.4 μm from Dr. Maisch) with a 50 min gradient from 5 to 60% acetonitrile in 0.1% formic acid. The effluent from the HPLC was directly electrosprayed into a LTQ-Orbitrap mass spectrometer (Flag-Trnp1 vs Trnp1 Ref399) or a Q Exactive HF (Flag-delta 1-16_Trnp1 vs delta 1-16_Trnp1 Ref1644). For LTQ-Orbitrap measurements, the instrument was operated in data dependent mode to automatically switch between full scan MS and MS/MS acquisition. Survey full scan MS spectra (from m/z 300 – 2000) were acquired in the Orbitrap with resolution R=60,000 at m/z 400 (after accumulation to a ‘target value’ of 500,000 in the linear ion trap). The six most intense peptide ions with charge states between 2 and 4 were sequentially isolated to a target value of 10,000 and fragmented in the linear ion trap by collision induced dissociation (CID). All fragment ion spectra were recorded in the LTQ part of the instrument. For all measurements with the Orbitrap detector, 3 lock-mass ions from ambient air were used for internal calibration. Typical MS conditions were: spray voltage, 1.5 kV; no sheath and auxiliary gas flow; heated capillary temperature, 200ºC; normalized CID energy 35%; activation q = 0.25; activation time = 30 ms. For Q Exactive HF measurements, the mass spectrometer was operated operated in data dependent mode to automatically switch between full scan MS and MS/MS acquisition. Survey full scan MS spectra (from m/z 375–1600) were acquired with resolution R=60,000 at m/z 400 (AGC target of 3x106). The 10 most intense peptide ions with charge states between 2 and 5 were sequentially isolated to a target value of 1x105, and fragmented at 27% normalized collision energy. Typical mass spectrometric conditions were: spray voltage, 1.5 kV; no sheath and auxiliary gas flow; heated capillary temperature, 250°C; ion selection threshold, 33.000 counts. MaxQuant 1.5.2.8 was used to identify proteins and quantify by iBAQ with the following parameters: Database, Uniprot_Mmusculus_UP000000589_10090_151030; MS tol, 10ppm; MS/MS tol, 0.5 Da (Orbitrap LTQ) or 10ppm (QExactive); Peptide FDR, 0.1; Protein FDR, 0.01 Min. peptide Length, 5; Variable modifications, Oxidation (M); Fixed modifications, Carbamidomethyl (C); Peptides for protein quantitation, razor and unique; Min. peptides, 1; Min. ratio count, 2. Identified proteins were considered as interaction partners, if their MaxQuant iBAQ values displayed a greater than 2-fold change enrichment and p-value 0.05 (ANOVA) when compared to the control.