MassIVE MSV000088076

Complete Public PXD028229

The CP110-CEP97-CEP290 module orchestrates a centriolar satellite2 dependent response to proteotoxic stress

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Description

Generation of Flp-In T-REx 293 cells for miniTurbo proximity labelling Proximity labelling was carried out utilizing miniTurbo. Full-length CP110 cDNA was cloned into a pcDNA5-FRT/TO-miniTurbo-FLAG destination vector via the Gateway cloning system. Using the Flp-In system, HEK293 cells stably expressing miniTurbo-FLAG (control) and miniTurboFLAG-CP110 were generated. Sample preparation for mass spectrometry (MS) Each cell pellet was resuspended in 10 mL of RIPA lysis buffer and streptavidin sepharose beads (GE Healthcare) was added. Beads were washed and washed twice more with ammonium bicarbonate buffer and tryptic digestion was performed by incubating the beads with TPCK-treated modified trypsin (Promega, Madison, WI). Supernatants were transferred to a fresh Eppendorf tube. The samples were lyophilized and resuspended in buffer A (0.1% formic acid). Mass spectrometry High-performance liquid chromatography was conducted using a 2 cm pre-column and 50 cm analytical column was performed, running a 120-min reversed-phase buffer gradient at 225 nl/min on a Proxeon EASY-nLC 1000 pump in-line with a Thermo Q Exactive HF quadrupole-Orbitrap mass spectrometer. A parent ion scan was performed using a resolving power of 60,000, then up to the twenty most intense peaks were selected for MS/MS (minimum ion count of 1,000 for activation), using higher energy collision-induced dissociation (HCD) fragmentation. Dynamic exclusion was activated such that MS/MS of the same m/z (within a range of 10 ppm; exclusion list size = 500) detected twice within 5 s was excluded from analysis for 15 s. For protein identification, Thermo. RAW files were converted to the .mzXML format using ProteoWizard, then searched using X!Tandem and Comet against the human Human RefSeq Version 45 database (containing 36,113 entries). Search parameters specified a parent ion mass tolerance of10 ppm and an MS/MS fragment ion tolerance of 0.4 Da, with up to 2 missed cleavages allowed for trypsin. Variable modifications of +16(M W), +32 (M and W), +42 (Nterminus), and +1 (N and Q) were allowed. [doi:10.25345/C50G23] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Flp-In T-REx HEK293, miniTurbo, BioID, biotin, streptavidin, trypsin, LC-MS ; QEHF

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Principal Investigators:
(in alphabetical order) 		Brian Raught, Princess Margaret Cancer Centre, Canada
Submitting User: jonstgermain
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