Dataset Information

Mass spectrometry-based protein interactome and phosphoproteomics data upon NRDE2 overexpression

ABSTRACT: The HEK293T cells transiently expressing Flag-tagged empty vector or Flag-NRDE2-WT were lysed in NETN buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40) and immunoprecipitated with anti-Flag M2 agarose (M8823; Sigma-Aldrich, MO, USA). After washing four times with the high stringent lysis buffer, the immunoprecipitants were resolved on gradient SDS-PAGE, silver-stained, and subjected to MS sequencing and data analyses. In-solution and in-gel digestion were performed according to a previously published method (Jin et al., 2003). Briefly, gel bands were minced and destained with 50% acetonitrile in 50 mM ammonium bicarbonate. Proteins were reduced with 10 mM dithiothreitol (DTT) at 56°C, followed by alkylation with 55 mM iodoacetamide at room temperature in the dark. Trypsin digestion was performed overnight at 37°C with gentle shaking. Peptides were extracted using 1% trifluoroacetic acid in 50% acetonitrile. Samples were vacuum-dried and reconstituted in 0.1% formic acid for subsequent MS analysis. MS analysis was performed using an Easy-nLC 1200 UHPLC coupled to a QExactive HF-X mass spectrometer (Thermo Fisher Scientific, MA, USA). The tandem mass spectra were searched against the human UniProt database (Version 20140922; including 20,193 sequences) using MaxQuant (Version 1.5.3.30) (Tyanova et al., 2016). Trypsin was selected as the proteolytic enzyme, and two missed cleavages sites were allowed. Cysteine carbamidomethylation was set as the fixed modification. The oxidation of methionine (M) residues and acetylation of the protein N-terminal were set as the variable modifications. The first search mass tolerance was 20 parts-per-million (ppm), and the main search peptide tolerance was 4.5 ppm. The FDRs of the peptide-spectrum matches (PSMs) and proteins were set to less than 1%.

ORGANISM(S): Homo Sapiens

SUBMITTER: Gangqiao Zhou

PROVIDER: PXD051117 | iProX | Sun Mar 31 00:00:00 GMT 2024

REPOSITORIES: iProX

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Publications

NRDE2 deficiency impairs homologous recombination repair and sensitizes hepatocellular carcinoma to PARP inhibitors.

Wang Yahui Y Liu Xinyi X Zuo Xianbo X Wang Cuiling C Zhang Zheng Z Zhang Haitao H Zeng Tao T Chen Shunqi S Liu Mengyu M Chen Hongxia H Song Qingfeng Q Li Qi Q Yang Chenning C Le Yi Y Xing Jinliang J Zhang Hongxin H An Jiaze J Jia Weihua W Kang Longli L Zhang Hongxing H Xie Hui H Ye Jiazhou J Wu Tianzhun T He Fuchu F Zhang Xuejun X Li Yuanfeng Y Zhou Gangqiao G

Cell genomics 20240501 5

To identify novel susceptibility genes for hepatocellular carcinoma (HCC), we performed a rare-variant association study in Chinese populations consisting of 2,750 cases and 4,153 controls. We identified four HCC-associated genes, including NRDE2, RANBP17, RTEL1, and STEAP3. Using NRDE2 (index rs199890497 [p.N377I], p = 1.19 × 10<sup>-9</sup>) as an exemplary candidate, we demonstrated that it promotes homologous recombination (HR) repair and suppresses HCC. Mechanistically, NRDE2 binds to the s ...[more]

PMID: 38697125

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Project description:1ml of WT, delta-pka, delta-arcA and delta-pka delta-arcA culture broth was harvested at u=0.4 h-1, centrifuged (14000 x g for 1 min at 4 degrees C), pellet washed once with PBS, flash frozen with liquid N2 and kept at -80 degrees C until further processing. Frozen pellets were melted on ice after which 100 ug of sample biomass was mixed with 100 ug of SILAC-labeled E. coli biomass (internal standard) and frozen at -80 degrees C. Cell pellets were suspended in 100 uL SDS lysis buffer (4% SDS/100 mM TRIS-HCl pH 8/100 mM DTT) and heated at 95 degrees C for 15 min. Cell lysates were sonicated with ultrasound for a few pulses and pelleted by centrifugation. Cell lysates were digested with trypsin according to Filter Aided Sample Preparation protocol (FASP) (Wiśniewski et al. 2009) and purified with C-18 StageTips (Rappsilber et al. 2007). LC-MS/MS analysis was performed using an Agilent 1200 series nanoflow system (Agilent Technologies) connected to a LTQ Orbitrap mass-spectrometer (Thermo Electron, San Jose, CA, USA) equipped with a nanoelectrospray ion source (Proxeon, Odense, Denmark). Peptides were separated with a 240-minute gradient from 2 to 40% B (A: 0.5% acetic acid, B: 0.5% acetic acid/80% acetonitrile) using a flow-rate of 200 nl/min. Data analysis of raw MS-files was performed by the MaxQuant software package version 1.3.0.5 (Cox and Mann 2008). Peak lists were searched using the Andromeda search engine (built into MaxQuant) against an E. coli database (downloaded 09.04.2013 from http://www.uniprot.org/) which was supplemented with common contaminants (e.g human keratin, trypsin). Full tryptic specificity, a maximum of two missed cleavages and a mass tolerance of 0.5 Da for fragment ions was specified in the MaxQuant search. Carbamidomethylation of cysteine was set as a fixed modification, and methionine oxidation and protein N-terminal acetylation were set as variable modifications. The required false discovery rate was set to 1% for both peptide and protein levels and the minimum required peptide length was set to six amino acids. “Match between runs” option with a time window of 1.5 min was allowed.

Project description:Proteomic analysis of chick vestibualr hair bundles purified using the twist-off method. See Description.doc. Utricle hair bundles were purified from E20–21 chicks using the twist-off method (Gillespie & Hudspeth J Cell Biol 112, 625-640, 1991; Shin et al. Neuron 53, 371-386, 2007). NuPAGE LDS sample buffer (Invitrogen) with 50 mM dithiothreitol was added to a combined final volume of 80 µl per 100 utricles' worth of bundles; samples were heated to 70°C for 15 min. Epithelial proteins were similarly solubilized with sample buffer. Proteins were separated by running ~1 cm into a NuPAGE 4–12% Bis-Tris gel (1.5 mm x 10 well; one or two lanes per bundle sample); gels were rinsed with water, then stained with Imperial Protein Stain (Thermo Scientific). The 1 cm of gel with separated proteins was manually sliced into six pieces. Gel pieces were transferred to siliconized tubes and washed and destained in 200 µl 50% methanol overnight. The gel pieces were dehydrated in acetonitrile, rehydrated in 30 µl of 10 mM dithiothreitol in 0.1 M ammonium bicarbonate and reduced at room temperature for 0.5 h. The DTT solution was removed and the sample alkylated in 30 µl 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 0.5 h. The reagent was removed and the gel pieces dehydrated in 100 µl acetonitrile. The acetonitrile was removed and the gel pieces rehydrated in 100 µl 0.1 M ammonium bicarbonate. The pieces were dehydrated in 100 µl acetonitrile, the acetonitrile removed and the pieces completely dried by vacuum centrifugation. The gel pieces were rehydrated in 20 ng/µl trypsin (Sigma-Aldrich T6567 proteomics grade, from porcine pancreas, dimethylated) in 50 mM ammonium bicarbonate on ice for 10 min. Any excess enzyme solution was removed and 20 µl 50 mM ammonium bicarbonate added. The sample was digested overnight at 37°C and the peptides formed extracted from the polyacrylamide in two 30 µl aliquots of 50% acetonitrile/5% formic acid. These extracts were combined and evaporated to 15 µl for MS analysis. For the gel slice immediately adjacent to the agarose, peptides were purified away from interfering polymers by SCX. A single experiment's worth of hair bundles or epithelium was analyzed by six LC-MS/MS runs, corresponding to the six gel pieces. The LC-MS/MS system consisted of a Thermo Electron Orbitrap Velos ETD mass spectrometer system with a Protana nanospray ion source, which was interfaced to a reversed-phase capillary column of 8 cm length x 75 µm internal diameter, self-packed with Phenomenex C18 Jupiter of 10 µm particle size. An extract aliquot (7.5 µl) was injected and peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.5 µl/min over 1.2 hr. The nanospray ion source was operated at 2.5 kV. The digest was analyzed using the data-dependent capability of the instrument, acquiring—in sequential scans—a single full scan mass spectrum in the Orbitrap detector at 60,000 resolution to determine peptide molecular weights, and 20 product-ion spectra in the ion trap to determine amino acid sequence. MaxQuant version 1.2.2.5 software was used for protein identification and quantitation. The default contaminants file associated with the MaxQuant download was edited to remove entries known to be present in hair bundles (e.g., actin) and to add additional impurities that entered the bundle-purification workflow (keratins, hemoglobins, egg white components). Mass spectrometry data were searched against Ensembl version 66 (released February, 2012) using Andromeda; the Ensembl FASTA file was edited by replacing several sequences with longer or full-length sequences, including actin gamma 1 (NP_001007825.1), actin beta (NP_990849.1), fascin 1 (NP_001171603), fascin 2 (NP_001171209), ATP synthase beta (NP_001026562.1), peptidylprolyl isomerase A (NP_001159798.1), calbindin 2 (NP_990647.1), PDZD7 (XP_003641537.1), espin (XP_417532.3), and CACNA2D2 (XP_427707.3). Protein identifications were reported with an FDR of 1%. If a set of peptides for a protein was identical to or completely contained within that of another protein, MaxQuant groups those proteins together ("redundant groups"); the entry with the largest number of peptides was used to identify the redundant group. Redundant groups that shared more than 20% of their identified peptides were further grouped in our analysis ("shared-peptide groups"); the entry with the greatest intensity associated with it was used to identify the shared-peptide group.

Project description:Serum samples were inactivated and sterilized at 56 ℃ for 30 min and processed as previous study with some modifications (Shen et al., 2020). Briefly, 4 μL serum from each sample was depleted using High Select Top-14 Abundant Protein Depletion MiniSpin Columns (Thermo Fisher Scientific, San Jose, USA). Then the elutes were denatured, reduced and alkylated to derive protein lysates. The solutions were diluted with 200 μL 100 mM triethylammonium bicarbonate (TEAB) followed by a double-step trypsinization (enzyme-to-substrate ratio kept at 1:40 in each step). 10% trifluoroacetic acid (TFA) was added to each sample to quench the enzymatic reaction. Digested peptides were cleaned-up with SOLAμ (Thermo Fisher Scientific, San Jose, USA) according to the manufacturer’s instructions and labeled by TMTpro 16 plex reagents (Thermo Fisher Scientific, San Jose, USA). 660 samples in total (633 serum samples and 27 technical replicates) were divided into 44 batches for labelling experiment, each batch containing fifteen samples and one pool. The labeled samples in each batch were combined and fractionated. Each sample was separated into 30 fractions and then consolidated into 10 fractions. Dried and re-dissolved fractions with 2% acetonitrile (ACN)/0.1% formic acid (FA) of MS grade. The re-dissolved peptides were analyzed with a U3000 HPLC system coupled to a Orbitrap Exploris 480 (Thermo Fisher Scientific, Bremen, Germany) in data-dependent acquisition (DDA) mode combined to a front-end high-field asymmetric waveform ion mobility spectrometry (FAIMS). Peptides of each fraction were loaded onto a pre-column (3 μm, 100 Å, 20 mm*75 mm i.d.) and separated with a 75 min LC gradient at a flow rate of 300 nL/min (analytical column: 1.9 mm, 120 Å, 150 mm*75 mm i.d.; Buffer A: 2% ACN, 98% H2O containing 0.1% FA; Buffer B: 98% ACN, 2% H2O containing 0.1% FA). FAIMS was operated at two different CV, -48 V and -68 V, respectively. For MS acquisition, RF level was set at 50% and ion transfer tube temperature at 320 °C. The turbo-TMT mode was enabled. Scan range of MS1 was 350–1800 m/z. Resolutions were set to 60000 for MS1 and 30000 for MS2. Normalized AGC target was set at 300% for MS1 and 200% for MS2. The maximum injection time was set as 50 ms for MS1 and 86 ms for MS2. Dynamic exclusion was on and mass tolerance was set to ±10 ppm. Intensity threshold was set at 20000 for MS1, and HCD collision energy was fixed to 38%. MS/MS data was recorded in centroid mode. Isolation window was set to 0.7 m/z.

Dataset Information

Mass spectrometry-based protein interactome and phosphoproteomics data upon NRDE2 overexpression

Publications

NRDE2 deficiency impairs homologous recombination repair and sensitizes hepatocellular carcinoma to PARP inhibitors.

Similar Datasets

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