Dataset Information

MudPIT analyses of the proteins co-purified with the Interactor of Little Elongation Complex ELL subunit 1 (FLAG-ICE1), WT MED26, and D2G8 NTD-mutant MED26, affinity-purified from HEK293T cell lines

ABSTRACT: Protein Complexes Purification: We generated 293FRT cell lines that stably express FLAG-tagged ICE1 (Interactor of Little Elongation Complex ELL subunit 1; KIAA0947). We extracted nuclear proteins in the presence of Benzonase to digest nucleic acids, including both DNA and RNA, and purified the little elongation complex (LEC) through anti-FLAG affinity purification using anti-FLAG M2 agarose. Cells only expressing the FLAG epitope-tag were analyzed in parallel as negative controls. A MED26 hypomorphic HEK293T cell line D2G8 was generated by applying multiple small guide RNAs (sgRNAs) complementary to the MED26 gene to induce random sequence insertions and/or deletions. This cell line expresses mutant MED26 lacking the NTD. Mediator was purified from cells expressing either WT MED26 or D2G8 MED26 via its ability to bind the transcriptional activation domain of ATF6-alpha. Proteins were precipitated with 1/5 TCA overnight at 4oC and washed 2x with cold acetone. Multidimensional Protein Identification Technology: TCA-precipitated protein pellets were with Tris-HCl pH 8.5 8 M urea, followed by addition of TCEP (Pierce) and chloroacetamide (Sigma) to a final concentration of 5 mM and 10 mM, respectively. Proteins were digested using Endoproteinase Lys-C (Roche) at 37oC overnight. The samples were brought to a final concentration of 2 M urea and 2 mM CaCl2 and a second digestion was performed overnight at 37oC using trypsin (Promega). Digested peptides were loaded on a split-triple-phase fused-silica micro-capillary column and placed in-line with a linear ion trap mass spectrometer (LTQ, Thermo Scientific), coupled with a Quaternary Agilent 1100 Series HPLC system. A fully automated 10-step chromatography run was carried out. Each full MS scan (400-1600 m/z) was followed by five data-dependent MS/MS scans. The number of the micro scans was set to 1 both for MS and MS/MS. The settings were as follows: repeat count 2; repeat duration 30 s; exclusion list size 500 and exclusion duration 120 s, while the minimum signal threshold was set to 100. MS Data Processing: The MS/MS data set was searched using ProLuCID (v. 1.3.3) against 36628 non-redundant Homo sapiens proteins (downloaded from NCBI RefSeq 2016-06-10), 193 usual contaminants, and, to estimate false discovery rates (FDRs), 36821 randomized amino acid sequences derived from each NR protein. To account for alkylation by CAM, 57 Da were added statically to the cysteines. To account for oxidation, 16 Da were added as a differential modification to methionines. Peptide/spectrum matches were sorted and selected to an FDR less than 5% at the peptide and protein levels, using DTASelect in combination with swallow, an in-house software.

INSTRUMENT(S): LTQ

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

SUBMITTER: Laurence Florens

PROVIDER: MSV000083465 | MassIVE | Wed Feb 20 13:47:00 GMT 2019

SECONDARY ACCESSION(S): PXD012807

REPOSITORIES: MassIVE

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Publications

The role of Mediator and Little Elongation Complex in transcription termination.

Takahashi Hidehisa H Ranjan Amol A Chen Shiyuan S Suzuki Hidefumi H Shibata Mio M Hirose Tomonori T Hirose Hiroko H Sasaki Kazunori K Abe Ryota R Chen Kai K He Yanfeng Y Zhang Ying Y Takigawa Ichigaku I Tsukiyama Tadasuke T Watanabe Masashi M Fujii Satoshi S Iida Midori M Yamamoto Junichi J Yamaguchi Yuki Y Suzuki Yutaka Y Matsumoto Masaki M Nakayama Keiichi I KI Washburn Michael P MP Saraf Anita A Florens Laurence L Sato Shigeo S Tomomori-Sato Chieri C Conaway Ronald C RC Conaway Joan W JW Hatakeyama Shigetsugu S

Nature communications 20200226 1

Mediator is a coregulatory complex that regulates transcription of Pol II-dependent genes. Previously, we showed that human Mediator subunit MED26 plays a role in the recruitment of Super Elongation Complex (SEC) or Little Elongation Complex (LEC) to regulate the expression of certain genes. MED26 plays a role in recruiting SEC to protein-coding genes including c-myc and LEC to small nuclear RNA (snRNA) genes. However, how MED26 engages SEC or LEC to regulate distinct genes is unclear. Here, we ...[more]

PMID: 32102997

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Project description:Proteomic analysis of pervanadate-induced tyrosine-phosphorylated proteins in hepatocellular carcinoma WRL 68 cells Protein tyrosine phosphorylation plays critical roles in modulating biological processes such as cellular proliferation, differentiation, migration, apoptosis and metabolism. To profile tyrosine phosphorylated (pTyr) proteins as well as search novel pTyr proteins as cross-talk points among different cellular pathways, we developed a rapid and efficient approach to identify cellular pTyr proteins and their complexes by a combination of subcellular proteomics approach with signal transduction strategies. Human hepatocytic cells from WRL68 cell line were treated with pervanadate (POV), subfractionated into four fractions and then subjected to immunoaffinity purification with anti-pTyr antibody. The eluted mixtures of the anti-pTyr purification were identified by LC-MS/MS. Subcellular fractionation and affinity purification of tyrosine-phosphorylated proteins: WRL68 cells were first grown to 80% confluence in MEM complete medium and then the medium replaced with serum free media. After 15 h, the cells were either untreated or stimulated with 0.1 mM pervanadate (1 mM sodium orthovanadate, 3 mM H2O2) for 10 min. 150-mm cultures of WRL 68 cells were rinsed twice with 4? PBS and then scraped from the dish in 750?l of hypotonic buffer (10 mM Tris, 1 mM NaF, 10 mM IAA, pH 7.5) containing a cocktail of protein inhibitors. After a 20-min incubation on ice, the cells were passed about five times through a 25-g needle. The resulting lysate was subjected to a 15min centrifugation at 1000 rpm at 4?, after which the pellet was resuspended in 250?l of hypotonic buffer and re-extracted by a second round of the trituration and centrifugation. The supernatants of the first and second spins were combined, adjusted to 0.25 M NaCl, and separated into cytosolic (supernatant) and membrane (pellet) fractions bycentrifugation at 19,000 rpm (43,000 g) for 90 min at 4?. All the pellets and total cell lysate were resuspended in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid sodium) containing 1mM pervanadate with a cocktail of protein inhibitors with sonication aid. Cleared cell lysates were incubated overnight at 4? with 30?l monoclonal anti-phosphotyrosine-agrose (Sigma). Precipitated immune complexes were washed three times with 1×HNTG (20 mM HEPES, 150 mM NaCl, 0.1% Triton X-100, 10% Glycerol, pH 7.5) and then eluted with 100 mM phenyl phosphate (Sigma) in lysis buffer at 4?. Enzyme digestion, mass spectrometry and protein identification: The sample was digested according to the published method. Chromatography was performed using a surveyor LC system (Thermo Finnigan,SanJose,CA) on C18 reverse phase column(RP, 180 µm x 150 mm, BioBasic® C18, 5 µm, Thermo Hypersil-Keystone). The pump flow rate is split 1:100 for a colum flow rate of 1.5 µL/min.The column effluent is directly electrosprayed using the orthogonal metal needle source without further splitting.Mobile phase A is 0.1% formic acid in water,and the B mobile phase is 0.1% formic acid in acetonitrile. The separation of peptides obtained by enzymatic digest of bile sample was achieved with a gradient of 2-80% B over 360 min.The column effluent from the reverse phase column was analyzed by LCQ Deca XP ion-trap mass spectrometer.The micro-electrospray interface uses a 30 µm metal needle that is orthogonal to the inlet of the LCQ.The mass spectrometer was set that one full MS scan was followed by three MS/MS scans on the three most intense ion from the MS spectrum with the following Dynamic Exclusion™ settings: repeat count, 1; repeat duration, 0.5 min; exclusion duration, 3.0 min. The acquired MS/MS spectra were automatically searched against protein database for human proteins (SWISS-PROT/TrEMBL) using the TurboSEQUEST program in the BioWorks™ 3.0 software suite. An accepted SEQUEST result had to have a ?Cn score of at least 0.1 (regardless of charge state) and Xcorr (one charge?1.5, two charges?2.0, three charges?2.5). Single peptides that alone identify a protein were manually validated after meeting the above criteria. Bioinformatics analysis: The pI and MW of the proteins were analyzed using ExPASy proteomics tools accessed from http://cn.expasy.org/tools/#proteome. The grand average hydropathicity (GRAVY) values were determined according to Kyte-Doolittle. The protein subcellular location annotation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/). The protein function family was categorized according to Gene Ontology (GO) annotation terms extracted by InterPro (http://www.ebi.ac.uk./interpro/). The annotation of protein phosphorylation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/) and PhosphoSite (http://www.phosphosite.org). The kinases were annotated according to human kinome. Keywords: other

Project description:Protein pull-down assay- Mixed erythrocytic stage 3D7 Plasmodium falciparum parasite cultures were collected and lysed using 0.15% saponin for 10 min on ice. After subsequent washes, the parasite pellet was resuspended in 2.5X volume of IP buffer (0.65% Igepal CA-360 (Sigma-Aldrich), 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton-X, 1 mM AEBSF, 5 uM E-64 and EDTA-free protease inhibitor cocktail (Roche)) and lysed by passing through a 26G 1/2-inch needle ten times and sonicated 7 times 10 seconds on/30 seconds off using a probe sonicator. Extracted nuclear protein lysates were incubated for 10 mins at room temperature with DNase I to remove DNA and centrifuged for 10 mins at 14,500 rcf to remove cell debris. Washed Protein A magnetic beads (Pure Proteome) were added to the protein sample and incubated for 1 hour at 4oC to preclear the lysate. Precleared lysate was transferred to a new microcentrifuge tube and split equally for the antibody and no antibody control. The anti-CRWN-like custom antibody was added at a 1:50 ratio and incubated overnight at 4oC. The negative control with no antibody was also incubated overnight. Antibody-protein complexes were recovered using Protein A magnetic beads (Pure Proteome), followed by extensive washes with wash buffer A (1% Triton-X, 1 mM EDTA in 1X PBS), wash buffer B (wash buffer A, 0.5 M NaCl) and wash buffer C (1 mM EDTA, 1X PBS). Proteins were eluted using 0.1 M glycine, pH 2.8 and the eluent was neutralized using 2 M Tris-HCl, pH 8.0. Protein eluates were TCA-precipitated overnight, then washed twice with cold acetone. Multidimensional Protein Identification Technology- The TCA-precipitated protein pellet was solubilized using Tris-HCl pH 8.5 and 8 M urea, followed by addition of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride; Pierce) and CAM (chloroacetamide; Sigma) were added to a final concentration of 5 mM and 10 mM, respectively. Proteins were digested using Endoproteinase Lys-C at 1:100 w/w (Roche) at 37oC overnight. The samples were brought to a final concentration of 2 M urea and 2 mM CaCl2 and a second digestion was performed overnight at 37oC using trypsin (Promega) at 1:100 w/w. The reactions were stopped using formic acid (5% final). The samples were loaded on a split-triple-phase fused-silica micro-capillary column and placed in-line with a linear ion trap mass spectrometer (LTQ, Thermo Scientific), coupled with a Quaternary Agilent 1100 Series HPLC system. A fully automated 10-step chromatography run was carried out. Each full MS scan (400-1600 m/z) was followed by five data-dependent MS/MS scans. The number of the micro scans was set to 1 both for MS and MS/MS. The settings were as follows: repeat count 2; repeat duration 30 s; exclusion list size 500 and exclusion duration 120 s, while the minimum signal threshold was set to 100. The MS/MS data set was searched using ProLuCID (v. 1.3.3) against a database consisting of 5,530 P. falciparum non-redundant proteins (PlasmoDB-34), 36,628 Homo sapiens NR proteins (NCBI -released June 10, 2016), 193 usual contaminants, and, to estimate false discovery rates (FDRs), 42,351 randomized amino acid sequences derived from each NR protein entry. To account for alkylation by CAM, 57 Da were added statically to the cysteine residues. To account for the oxidation of methionine to methionine sulfoxide, 16 Da were added as a differential modification to the methionine residue. Peptide/spectrum matches were sorted and selected to an FDR less than 5% at the peptide and protein levels, using DTASelect in combination with swallow, an in-house software.

Project description:Mammalian protein extraction and Halo purification For Halo purification, HEK293T cells at 40% confluency in a 100 mm plate were transfected with 10ug of plasmid containing ORF of PDHA1-HA-Halo in 1X BBS with 125 mM CaCl2. Cells were harvested by scraping in PBS following PBS wash after 24 hours from transfection. For non-transfected cells, cells were harvested at 100% confluency. The cell pellet was resuspended in 1 ml of Hypotonic buffer (50 mM Tris-HCl pH 7.5, 0.1% NP-40) and centrifuged at 4000 rpm for 5 min. The resulting pellet was resuspended in 1 ml of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 % Triton X-100, 0.1 % Na-deoxycholate) and clarified by centrifugation at 12,000 rpm for 30 min. The crude extract in the supernatant was treated with 125 U of benzonase for 20 minutes in 4oC, then clarified by centrifugation at 12,000 rpm for 10 minutes. The clarified extract was either directly loaded onto Superose 6 column or subjected to Halo-purification by adding with 100 ul of Halo beads suspension incubated overnight in 4oC on a rotator. The beads were washed 3 times then subjected for protein elution with 50 U of AcTEV protease (Invitrogen, 12575-015) in PBS buffer by 8 hr incubation in 4oC on a rotator. Multidimensional Protein Identification Technology Proteins isolated from Halo-AP of N- and C-terminally tagged human PDHA1 (Halo-E1-alpha and E1-alpha-Halo) were precipitated with 20% trichloroacetic acid (TCA) and the resulting pellet was washed once with 10% TCA and twice with cold acetone. TCA-precipitated protein pellet was solubilized using Tris-HCl pH 8.5 and 8 M urea, followed by addition of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride; Pierce) and CAM (chloroacetamide; Sigma) were added to a final concentration of 5 mM and 10 mM, respectively. Proteins were digested using Endoproteinase Lys-C at 1:100 w/w (Roche) at 37oC overnight. The samples were brought to a final concentration of 2 M urea and 2 mM CaCl2 and a second digestion was performed overnight at 37oC using trypsin (Promega) at 1:100 w/w. The reactions were stopped using formic acid (5% final). The samples were loaded on a split-triple-phase fused-silica micro-capillary column and placed in-line with a linear ion trap mass spectrometer (LTQ, Thermo Scientific), coupled with a Quaternary Agilent 1100 Series HPLC system. A fully automated 10-step chromatography run was carried out. Each full MS scan (400-1600 m/z) was followed by five data-dependent MS/MS scans. The number of the micro scans was set to 1 both for MS and MS/MS. The settings were as follows: repeat count 2; repeat duration 30 s; exclusion list size 500 and exclusion duration 120 s, while the minimum signal threshold was set to 100. The MS/MS data set was searched using ProLuCID (v. 1.3.3) against a database consisting of 36,628 Homo sapiens NR proteins (NCBI -released June 10, 2016), 193 usual contaminants, and, to estimate false discovery rates (FDRs), randomized amino acid sequences derived from each NR protein entry. To account for alkylation by CAM, 57 Da were added statically to the cysteine residues. To account for the oxidation of methionine to methionine sulfoxide, 16 Da were added as a differential modification to the methionine residue. Peptide/spectrum matches were sorted and selected to an FDR less than 5% at the peptide and protein levels, using DTASelect v1.9 in combination with swallow, an in-house software.