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

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Brian Raught

PROVIDER: MSV000088076 | MassIVE |

SECONDARY ACCESSION(S): PXD028229

REPOSITORIES: MassIVE

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Project description:Flp-In 293 T-REx cells (Thermo Fisher Scientific) stably expressing FlagBirA alone or FlagBirA-LIMP-2 were generated. After selection , cells were incubated for 24 hours in complete media supplemented with tetracycline (Sigma) and biotin (BioShop). Cells were collected and pelleted, the pellets were washed twice with PBS and snap frozen. Cells were resuspended in 10 ml of modified RIPA lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% SDS, 1:500 protease inhibitor cocktail (Sigma-Aldrich), 1:1,000 benzonase nuclease (Novagen), incubated on an end-over-end rotator , sonicated then centrifuged. Supernatants were transferred to a fresh 15 ml conical tube. Streptavidin-sepharose beads (GE) were added and the mixture incubated. Beads were pelleted by centrifugation at 2,000 rpm for 2 minutes and transferred with 1 ml of lysis buffer to a fresh Eppendorf tube. Beads were washed once with 1 mL lysis buffer and twice with 1 ml of 50 mM ammonium bicarbonate (pH =8.3). Beads were transferred in ammonium bicarbonate to a fresh centrifuge tube and washed two more times with 1 ml ammonium bicarbonate buffer. Tryptic digestion was performed by incubating the beads with MS-grade TPCK trypsin (Promega, Madison, WI) dissolved ammonium bicarbonate (pH 8.3). The following morning, additional MS-grade TPCK trypsin was added, and beads were incubated 2 additional hours. Beads were pelleted by centrifugation at 2,000 x g for 2 min, and the supernatant was transferred to a fresh Eppendorf tube. Beads were washed twice with 50 mM ammonium bicarbonate, and these washes were pooled with the first eluate. The sample was lyophilized and resuspended in buffer A (0.1% formic acid). 1/5th of the sample was analyzed per MS run. Resuspended peptides were subjected to high performance liquid chromatography (HPLC) performed on pre-columns (150mm I.D.) and analytical columns (75mm I.D.) assembled in-house using fused silica capillary tubing (InnovaQuartz, Phoenix, AZ) packed with C18 (Magic, Michrom Bioresources, Auburn, CA). A 120min reversed-phase LC gradient (0-100% organic, 0.1% HCOOH) running at 250 nl/min on a Proxeon EASY-nLC pump was applied in-line with a hybrid LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA). A parent ion scan was performed in the Orbitrap at 60,000 resolution (fwhm), with up to the 20 most intense precursors selected for MS/MS (minimum ion count of 1000 for activation), using standard collision-induced dissociation (CDI) fragmentation. Dynamic exclusion was activated such that MS/MS of the same m/z (within a range of 15 ppm; exclusion list size = 500) detected twice within 15 seconds were excluded from analysis for 30 sec. For protein identification, Thermo .RAW files were converted to the .mzXML format using Proteowizard (PMID 24939128), then searched using X!Tandem (PMID 14976030) against the human database (Human RefSeq Version 45), using a parent ion mass tolerance error of 15ppm and fragment ion mass of 0.4 Da. Oxidation (M), acetylation (protein N-terminus) and deamidation (N, Q) were included as potential modifications. Each biological replicate was analyzed using two technical replicates. Data were analyzed using the trans-proteomic pipeline PMID 16729052) via the ProHits software suite (PMID 20944583). Proteins identified with a Protein Prophet cut-off of 0.9 were analyzed with the SAINT (PMID 21131968) algorithm using 14 Flag-BirA only samples as negative controls. High confidence interactors were defined as those with a SAINT score over 0.8 (bayesian false discovery rate (BFDR) lower than 0.01).

Project description:Biochemical purification: Five grams of adult flies or 2.5 g of embryos were used in each affinity purification. Flies or embryos were suspended in 15 ml of buffer B (buffer A plus 1.5 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonylfluoride [PMSF], 0.5 5g/ml leupeptin, 0.8 5g/ml pepstatin, 20 U/ml DNase I, 100 U/ml RNasin [Promega], and 0.2 mg/ml heparin) in a mortar filled with liquid nitrogen and ground with a pestle to a fine powder. The powder was transferred to a glass-dounce, thawed and dounced until the pestle reached the bottom. The suspension was centrifuged twice at 40C and 10,000 g for 10 min. The fat layer on top was aspirated off after each centrifugation. Cleared extract (12.5 ml) was incubated with 600 5l of a slurry (50% [v/v]) of IgG-agarose beads (Sigma) for 90 min at 4C. The beads were washed once with buffer B for 15 min at 4C, and three times for 15 min at 4C with buffer C (20 mM TrisHCl [pH 8.0], 150 mM NaCl, 1 mM EDTA [pH 8.0], 10% glycerol, 0.01% NP-40, 1 mM DTT, 10 U/ml RNasin). PumHD was released from beads by incubation with 150 Units of AcTEV protease (Invitrogen) for 2 hr at room temperature (RT). RNA was isolated from extracts and from the TEV eluates with TRIZOL reagent (Invitrogen) followed by RNeasy Mini-Kit (Qiagen) purification according to the manufacturer's instructions. Microarray analysis: A pool of four control cDNAs (2 ng of each flp, gal4, lacZ and -Gal) was added to each RNA sample prior to labeling as controls for the labeling procedure. Total RNA (12 5g) derived from the extract and 300 ng or 30% of the affinity-isolated RNA were labeled with Cy3 and Cy5 fluorescent dyes, respectively, following cDNA synthesis with amino-allyl dUTP in addition to the four natural dNTPs using a 1:1 mixture of oligo(dT) and random nonamer primers. The Cy3- and Cy5-labeled cDNA samples were mixed and hybridized to Drosophila DNA microarrays. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Keywords: RNA IP

Project description:Biochemical purification: Five grams of adult flies or 2.5 g of embryos were used in each affinity purification. Flies or embryos were suspended in 15 ml of buffer B (buffer A plus 1.5 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonylfluoride [PMSF], 0.5 5g/ml leupeptin, 0.8 5g/ml pepstatin, 20 U/ml DNase I, 100 U/ml RNasin [Promega], and 0.2 mg/ml heparin) in a mortar filled with liquid nitrogen and ground with a pestle to a fine powder. The powder was transferred to a glass-dounce, thawed and dounced until the pestle reached the bottom. The suspension was centrifuged twice at 40C and 10,000 g for 10 min. The fat layer on top was aspirated off after each centrifugation. Cleared extract (12.5 ml) was incubated with 600 5l of a slurry (50% [v/v]) of IgG-agarose beads (Sigma) for 90 min at 4&#61616;C. The beads were washed once with buffer B for 15 min at 4&#61616;C, and three times for 15 min at 4&#61616;C with buffer C (20 mM TrisHCl [pH 8.0], 150 mM NaCl, 1 mM EDTA [pH 8.0], 10% glycerol, 0.01% NP-40, 1 mM DTT, 10 U/ml RNasin). PumHD was released from beads by incubation with 150 Units of AcTEV protease (Invitrogen) for 2 hr at room temperature (RT). RNA was isolated from extracts and from the TEV eluates with TRIZOL reagent (Invitrogen) followed by RNeasy Mini-Kit (Qiagen) purification according to the manufacturer's instructions. Microarray analysis: A pool of four control cDNAs (2 ng of each flp, gal4, lacZ and &#61538;-Gal) was added to each RNA sample prior to labeling as controls for the labeling procedure. Total RNA (12 5g) derived from the extract and 300 ng or 30% of the affinity-isolated RNA were labeled with Cy3 and Cy5 fluorescent dyes, respectively, following cDNA synthesis with amino-allyl dUTP in addition to the four natural dNTPs using a 1:1 mixture of oligo(dT) and random nonamer primers. The Cy3- and Cy5-labeled cDNA samples were mixed and hybridized to Drosophila DNA microarrays. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Keywords: RNA IP all_pairs

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

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