Shotgun proteomics (human)

For proteomic analysis, iPSC-derived neural stem cells at day 15 of culture were treated with 3.3 uM Nocodazole (Sigma) or same volume of DMSO (control) before being washed with ice-cold PBS, scrapped and centrifuged for 10 min at 300 g before lysis in Buffer A, containing 50 mM Tris-HCL (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-X100, 0.1% SDS, protease inhibitor (Pierce), at a volume twice that of the pellet. For neurons, day 40 of cultures treated only with DMSO were used. Samples were then stored on ice for cell lysis for 30 minutes before being transferred to protein low-bind tubes (Eppendorf) and centrifuged at 13,000 rpm for 10 min to pellet debris. Supernatant was collected and transferred to fresh protein low-bind tubes with protein quantified via DC Protein Assay (Bio-Rad). For each centrosomal bait protein immunoprecipitation 5 mg of protein was incubated with 2 ug of each respective antibody separately. Samples were rotated end-to-end for 1 hour at 4°C before 10 ul of each of the Protein A and Protein G Dynabeads (Invitrogen) were added. Samples were then further rotated end-to-end for 2 hours at 4°C before washed 3 times in lysis buffer. Samples were then incubated in 25 ul of Laemmli buffer (pH 6.8 with 1% SDS) and boiled at 95°C for 10 min and the eluate was stored at -80°C until LC-MS/MS or Western blot analysis. For PRPF6 immunoprecipitations, 1 ml of purified monoclonal 6C7 antibody at 10 ug/ml was incubated with Protein G Dynabeads (Invitrogen) with end-to-end rotation at 4°C for 2 hours before being washed three times in lysis buffer, before being added at a final volume of 20 ul to 5 mg of iPSC-derived neural stem cell protein lysate isolated at day 15 of culture. At least four biological replicates for each bait (IP) were performed, and control samples were obtained using the same procedure, only excluding the primary antibody. Total protein eluates were proteolysed with trypsin by a modified filter aided sample preparation (FASP) as described (3, 4). LC-MS/MS analysis was performed on a QExactive HF mass spectrometer (ThermoFisher Scientific, Waltham, Massachusetts, USA) online coupled to an Ultimate 3000 RSLC nano-HPLC (Dionex, Sunnyvale, California, USA). Samples were automatically injected and loaded onto the C18 trap column and after 5 min eluted and separated on the C18 analytical column (Acquity UPLC M-Class HSS T3 Column, 1.8 um, 75 um x 250 mm; Waters, Eschborn, Germany) by a 95 min non-linear acetonitrile gradient at a flow rate of 250 nl/min. MS spectra were recorded at a resolution of 60,000 and after each MS1 cycle, the 10 most abundant peptide ions were selected for fragmentation. The submitted project is structured into three separate datasets: ONA11883: NSC (day 15) centrosome interactome UZF13667: Neuron (day 40) centrosome interactome ONA13056: PRPF6 interactome