Protein Identification in Tetrahymena thermophila cilia IEX fractions used for XL-MS
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Tetrahymena Thermophila (ncbitaxon:5911)
SUBMITTER: 
Edward Marcotte
PROVIDER: MSV000091710 | MassIVE | Thu Apr 13 14:53:00 BST 2023
SECONDARY ACCESSION(S): PXD050674
REPOSITORIES: MassIVE
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Project description:Eleven genome wide microarrays containing the predicted coding sequences (putative genes) for the ciliated protozoan Tetrahymena thermophila used to study gene expression in conjugation cells (C-0, C-15m, C-2, C-4, C-6, C-8, C-10, C-12, C-14, C-16, C-18). Combined these eleven microarrays with 50 microarrays described in Miao et al (2009) and other 6 microarrays, we constructed the Tetrahymena gene network (TGN) using three methods: the Pearson correlation coefficient, the Spearman correlation coefficient and the context likelihood of relatedness (CLR) algorithm. The accuracy and coverage of the three networks were evaluated using four conserved protein complexes in yeast, and the CLR network was found to be the best network, with a Z-score threshold 3.49. Then the TGN was partitioned, and 55 modules were found. In addition, analysis for the arbitrarily determined 1200 hubs showed that these hubs could be sorted into six groups according to expression profiles. We also investigated human disease orthologs in Tetrahymena that are missing in yeast and found evidence indicating that some of these were involved in the same process in Tetrahymena as in human. For conjugation, B2086 and CU428 cells that had been starved for 18 hours were resuspended in 10 mM Tris (pH 7.5) at 200,000 cells/ml, mixed, and samples were collected at 15 min, 2, 4, 6, 8, 10, 12, 14, 16, 18 hours after the mixture （referred to as C-0, C-15m, C-2, C-4, C-6, C-8, C-10, C-12, C-14, C-16 and C-18).
2011-06-13 | E-GEOD-26650 | biostudies-arrayexpress
Project description:Procedure details are reported in McCafferty et al. eLife2022;11e81977. Briefly cilia extract from Tetrahymena thermophila SB715 was fractionated on a mixed bed IEX HPLC column and all resulting fractions were crosslinked by addition of DSSO to 0.5mM. Following reduction, alkylation, trypsin digestion, and desalting, mass spectra were collected on an Orbitrap Fusion Lumos using a DDA MS2-MS3 method and spectra were processed in ProteomeDiscoverer 2.3 using the Xlinkx node to identify crosslinks. The look-up database for crosslink identification was generated from the same .RAW files using a standard Basic workflow to identify proteins present in the fractions.
2023-04-20 | MSV000091761 | MassIVE
Project description:Two genome-wide microarrays containing the predicted coding sequences (putative genes) for the ciliated protozoan Tetrahymena thermophila were used to study gene expression in starved cells (starvation 0 hour and starvation 24 hour). Combining these two microarrays with 50 microarrays described in Miao et al. (2009) and 15 other microarrays, we constructed the Tetrahymena gene network (TGN) using three methods: the Pearson correlation coefficient, the Spearman correlation coefficient and the context likelihood of relatedness (CLR) algorithm. The accuracy and coverage of the three networks were evaluated using four conserved protein complexes in yeast, and the CLR network was found to be the best network, with a Z-score threshold 3.49. Then the TGN was partitioned, and 55 modules were found. In addition, analysis for the arbitrarily determined 1200 hubs showed that these hubs could be sorted into six groups according to expression profiles. We also investigated human disease orthologs in Tetrahymena that are missing in yeast and found evidence indicating that some of these were involved in the same process in Tetrahymena as in human. For starvation, CU428 cells were starved at 2x10^5 cells/ml in 10 mM Tris (pH 7.5) for 0 and 24 hours (referred to as S-0 and S-24, respectively).
2011-06-13 | E-GEOD-26384 | biostudies-arrayexpress
Project description:Four genome wide microarrays containing the predicted coding sequences (putative genes) for the ciliated protozoan Tetrahymena thermophila used to study gene expression in starved cells (Starvation 0 hour and Starvation 9 hour, each two replicates). Combined these four microarrays with 50 microarrays described in Miao et al (2009, PMID: 19204800; GSE11300) and other 13 microarrays, we constructed the Tetrahymena gene network (TGN) using three methods: the Pearson correlation coefficient, the Spearman correlation coefficient and the context likelihood of relatedness (CLR) algorithm. The accuracy and coverage of the three networks were evaluated using four conserved protein complexes in yeast, and the CLR network was found to be the best network, with a Z-score threshold 3.49. Then the TGN was partitioned, and 55 modules were found. In addition, analysis for the arbitrarily determined 1200 hubs showed that these hubs could be sorted into six groups according to expression profiles. We also investigated human disease orthologs in Tetrahymena that are missing in yeast and found evidence indicating that some of these were involved in the same process in Tetrahymena as in human. For starvation, CU427 cells were starved at 2x10^5 cells/ml in 10 mM Tris (pH 7.5) for 0 and 9 hours (referred to as S-0 and S-9, respectively).
2011-06-13 | E-GEOD-26385 | biostudies-arrayexpress
Project description:A genome wide microarray platform containing the predicted coding sequences (putative genes) for the ciliated protozoan Tetrahymena thermophila is described, validated and used to study gene expression in growing, starved and conjugating cells. Of the ~27,400 predicted open reading frames in Tetrahymena, transcripts homologous to 5876 are not detectable in one or more of these life cycle stages, indicating that this organism does indeed contain a large number of functional genes. Transcripts from over 5000 predicted genes are expressed at levels 5X background and 95 genes are expressed at 250X background in all stages. Transcripts homologous to 94 predicted genes are specifically expressed and 155 more are highly up-regulated in growing cells, and 90 are specifically expressed and 616 are up-regulated during starvation. Strikingly, transcripts homologous to 1073 predicted genes are specifically expressed and 1753 are significantly up-regulated during conjugation. The patterns of gene expression during conjugation correlate well with the previously described developmental stages of meiosis, nuclear differentiation and DNA elimination. Genes encoding proteins known to be complexed show similar expression patterns, indicating that co-ordinate expression with putative genes of known function should identify new genes with related functions. Keywords: growth (three cell densities); Starvation (seven time points); Conjugation (ten time points) For growth, CU428 cells at low, medium and high cell densities (~100Kcells/ml, ~350Kcells/ml and ~1000Kcells/ml; referred to as L-l, L-m and L-h) were collected. For starvation, CU428 cells were starved at 2x105 cells/ml in 10 mM Tris (pH 7.5) for 3, 6, 9, 12, 15 and 24 hours ï¼referred to as S-0, S-3, S-6, S-9, S-12, S-15 and S-24. For conjugation, B2086 and CU428 cells that had been starved for 18 hours were resuspended in 10 mM Tris (pH 7.5) at 2x105 cells/ml, mixed, and samples were collected at 2, 4, 6, 8, 10, 12, 14, 16, 18 hours after the mixture ï¼referred to as C-0, C-2, C-4, C-6, C-8, C-10, C-12, C-14, C-16 and C-18). For each growing and starved Tetrahymena sample, hybridizations were performed on three independent microarrays (e.g. L1, L2 and L3; S1, S2 and S3). For analysis of conjugation, hybridizations were performed on two independent microarrays (e.g. C1 and C2). Each individual microarray represents a separate experiment; total RNA was isolated from independent cell cultures then independently labeled and hybridized.
2010-06-25 | E-GEOD-11300 | biostudies-arrayexpress
Project description:Cilia were isolated from Tetrahymena thermophila SB715 by pH shock. Soluble ciliary proteins were extracted in buffer containing 1% NP40 and fractionated by HPLC SEC or mixed bed IEX. All fractions were reduced/alkylated and digested with trypsin for LC-MS/MS on an Orbitrap Fusion using a DDA 75 minute top speed CID method. Data were processed using MS Blender.
2023-04-05 | MSV000091648 | MassIVE
Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.
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Project description:Sample preparation: P-AMPK+AMP, P-AMPK+ATPγS, and AMPK+ATPγS protein samples were crosslinked in HBST buffer (25 mM HEPES pH 7.5 at room temperature, 200 mM NaCL, and 1 mM TCEP). Non-crosslinked negative controls were generated using the same procedure without the addition of DSSO. Reactions were initiated by spiking 1 uL of 75 mM DSSO (Thermo-Fisher) in DMSO into a 50 uL solution containing 10 uM protein and mixing. The final molar ratio of crosslinker:protein was 150:1. The reactions were incubated at 25°C for 45 minutes prior to being quenched by the addition of Tris pH 8.0 to a final concentration of 50 mM. Each crosslink reaction was done in triplicate and the reaction replicates were pooled after quenching. 10 μL samples of the pooled samples were loaded for SDS-PAGE analysis with Coomassie staining to confirm the presence of crosslinked protein. The remaining 140 μL of crosslinked and non-crosslinked samples were then acetone precipitated at -20°C overnight and the protein was pelleted by centrifugation at 16,000 rcf for 5 minutes at 4°C. After decanting, the pellets were dried for 15 minutes at room temperature before being resuspended in 12.5 uL of resuspension buffer (50 mM ammonium bicarbonate, 8M urea, pH 8.0). ProteaseMAX (Promega) was added to 0.02% and the solutions were mixed on an orbital shaker operating at 400 RPM for 5 minutes. After resuspension, 87.5 uL of digestion buffer (50 mM ammonium bicarbonate, pH 8.0) was added. Cysteines in the protein samples were reduced and akylated as follows. DTT was added to 5 mM final concentration and the protein solutions were incubated at 56°C in an orbital shaker operating at 400 RPM and for 20 minutes. After reduction, 2.7 uL of 550 mM iodoacetamide was added and the solutions were incubated in the dark at room temperature for 15 minutes. Reduced and alkylated protein solutions were digested using trypsin at a ratio of 1:200 (w/w trypsin:protein) and incubating at 37°C. After overnight incubation at 37°C, the samples were acidified by the addition of trifluoroacetic acid (TFA, Thermo-Fischer) to 1%. Samples were then frozen and stored at -20°C until analysis.
2021-07-08 | PXD019017 | Pride
Project description:Tetrahymena thermophil SB715 were deciliated by pH shock, collected by centrifugation, washed once and flash frozen. Lysate was made by grinding in liquid nitrogen and resuspension in buffer containing 0.2% NP40 with pipetting. Lysate was clarified by sequential centrifugations including 1 hour 130,000 x g 4C. Clarified lysate was fractionated by HPLC SEC or diluted to reduce salts to 22mM NaCl prior to fractionation by mixed-bed IEX. Fractions were reduced/alkylated and digested with trypsin for DDA LC-MS/MS. Data from SEC fractions was collected on an Orbitrap Fusion Lumos using a 75 minute top speed HCD method. Data from IEX fractions was collected on an Orbitrap Fusion using a 75 minute Topspeed CID method.
2023-04-07 | MSV000091661 | MassIVE
Project description:Intact cilia were isolated from dibucaine-treated Tetrahymena thermophila and subjected to DSSO crosslinking. Following quenching of the DSSO, a soluble "membrane and matrix" extract was generated by the addition of buffer containing 1% NP-40. The insoluble axonemes were removed by centrifugation and the supernatant extract was digested with trypsin, and crosslinked peptides were enriched using size exclusion chromatography.
Crosslinks were identified using an MS2-MS3 data collection method and the Proteome Discoverer XlinkX node. Crosslink searching used a smaller fasta generated from analysis of the .RAW files and the Basic Sequest workflow.
2022-08-01 | MSV000090056 | MassIVE