Dataset Information

MudPIT analysis of the proteins associated with FLAG-Hoxa1 and FLAG-Hoxb1 purified from chromatin fractions isolated from mouse KH2ES

ABSTRACT: MudPIT Analysis Four biological replicates of FLAG-Hoxa1, and corresponding negative controls, and FLAG-Hoxb1, and corresponding negative controls, were prepared from a chromatin enriched fraction isolated from mouse KH2ES cells and subjected to FLAG affinity purification. The eluted proteins were TCA-precipitated and analyzed by MudPIT. TCA-precipitated proteins were urea-denatured, reduced, alkylated and digested with endoproteinase Lys-C (Roche) followed by modified trypsin (Promega). Peptide mixtures were loaded onto 250 um fused silica microcapillary columns packed with strong cation exchange resin (Luna, Phenomenex) and 5-um C18 reverse phase (Aqua, Phenomenex), and then connected to a 100 um fused silica microcapillary column packed with 5-um C18 reverse phase (Aqua, Phenomenex). Loaded microcapillary columns were placed in-line with a Quaternary Agilent 1100 series HPLC pump and a LTQ linear ion trap mass spectrometer equipped with a nano-LC electrospray ionization source (ThermoScientific, San Jose, CA). Fully automated 10-step MudPIT runs were carried out on the electrosprayed peptides. Tandem mass (MS/MS) spectra were interpreted using ProluCID (v. 1.3.3) against a database consisting of 78014 nonredundant Mus musculus proteins (NCBI, 2016-06-23 release), 193 usual contaminants (human keratins, IgGs, and proteolytic enzymes). To estimate false discovery rates (FDR)s, the amino acid sequence of each non-redundant protein entry was randomized to generate a virtual library. This resulted in a total library of 116008 non-redundant sequences against which the spectra were matched. All cysteines were considered as fully carboxamidomethylated (+57 Da statically added), while methionine oxidation was searched as a differential modification. DTASelect (v. 1.9) and swallow, an in-house developed software, were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here all controlled FDRs were less than 5%. All 16 data sets were contrasted against their merged data set, respectively, using Contrast v 1.9 and in house developed sandmartin v0.0.1. Our in-house developed software, NSAF7 v0.0.1, was used to generate spectral count-based label free quantitation results.

INSTRUMENT(S): LTQ

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Laurence Florens

PROVIDER: MSV000085127 | MassIVE | Fri Mar 20 11:50:00 GMT 2020

SECONDARY ACCESSION(S): PXD018127

REPOSITORIES: MassIVE

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Sample Preparation: To identify proteins that are recruited by Hoxb1 to two newly identified DNA motifs, namely Motif_2 and LONG2x, nuclear extracts from 24hrs Retinoic acid- and Doxycycline-induced KH2 cell line with inducible Hoxb1 were added to DNA fragments immobilized on magnetic beads. Five experimental conditions were used: motif_2 (15bp) DNA, long2X (45bp) DNA, Hoxb1-ARE DNA as positive control, and 2 negative controls, random DNA and beads-only. In all cases, proteins were incubated at 30C for 30min to allow binding, followed by 2 sequential washes with buffer containing 0.1mg/ml BSA. Proteins were eluted by 2% SDS, 50mM Tris-HCl pH8.8 at 70C for 10 min, then precipitated by 20% TCA. MudPIT Analysis TCA-precipitated proteins were urea-denatured, reduced, alkylated and digested with endoproteinase Lys-C (Roche) followed by modified trypsin (Promega). Peptide mixtures were loaded onto 250 um fused silica microcapillary columns packed with strong cation exchange resin (Luna, Phenomenex) and 5-um C18 reverse phase (Aqua, Phenomenex), and then connected to a 100 um fused silica microcapillary column packed with 5-um C18 reverse phase (Aqua, Phenomenex). Loaded microcapillary columns were placed in-line with a Quaternary Agilent 1100 series HPLC pump and a LTQ linear ion trap mass spectrometer equipped with a nano-LC electrospray ionization source (ThermoScientific, San Jose, CA). Fully automated 10-step MudPIT runs were carried out on the electrosprayed peptides. Tandem mass (MS/MS) spectra were interpreted using ProluCID (v. 1.3.3) against a database consisting of 61441 non-redundant M. musculus proteins (NCBI, 2020-11-03 release), 426 usual contaminants (human keratins, IgGs, and proteolytic enzymes). To estimate false discovery rates (FDR)s, the amino acid sequence of each non-redundant protein entry was randomized, resulting in a total search space of 123728 non-redundant sequences. All cysteines were considered as fully carboxamidomethylated (+57 Da statically added), while methionine oxidation was searched as a differential modification. DTASelect (v. 1.9) and swallow, an in-house developed software, were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here all controlled FDRs were less than 5%. All 16 data sets were contrasted against their merged data set, respectively, using Contrast v 1.9 and in house developed sandmartin v0.0.1. Our in-house developed software, NSAF7 v0.0.1, was used to generate spectral count-based label free quantitation results.

Project description:Approximately 10E9 cells for each cell line (FLAG-DYRK1A, FLAG-DYRK1A-K188R, and FLAG-controls) were collected and washed with PBS. Cells were swollen for 15 minutes in hypertonic buffer (Buffer A: 10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT) freshly supplemented with protease inhibitor cocktail (Sigma P8340). Swollen cells were dounced 20 times in a Wheaton dounce homogenizer till about 90% cells were lysed (as observed in a microscope). Lysate is then centrifuged at 25Kg for 20 min, to pellet Nuclei and cell debris. To the supernatant (S100) 0.11 volume of Buffer B (0.3 M HEPES pH 7.9, 1.4 M KCl and 0.03 M MgCl2) was added and cleared by centrifuging at 100Kg, 4C for 1 hour. The supernatant was treated as cytoplasmic extract, and Flag-affinity was performed by incubating Flag beads for 3-4 hours. Beads were then washed with Flag wash buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 300 mM NaCl, 10 mM KCl, 0.2% TritonX-100) 3 times, and then eluted 2 times in elution buffer (200ug/ml Flag peptide, 10 mM HEPES pH7.9, 0.1 M NaCl, 1.5 mM MgCl2, 0.05% TritonX-100. Trichloroacetic acid-precipitated protein mixtures from the Flag purifications were digested with endoproteinase Lys-C and trypsin (Roche) and analyzed by a 10-step MudPIT (Multidimensional Protein Identification Technology) on a LTQ linear ion trap mass spectrometer (Thermo Scientific) coupled to a Quaternary Agilent 1100 series HPLC pump and a nano-LC electrospray ionization source. Tandem mass (MS/MS) spectra were interpreted using ProluCID (10.1016/j.jprot.2015.07.001) against a database consisting of 44093 nonredundant human proteins (collated from Genome Reference Consortium Human Build 38 patch release 13 and NCBI RefSeq 2019-12-03 release), 426 usual contaminants (human keratins, IgGs, and proteolytic enzymes) and the shuffled sequences of each non-redundant entry to estimate false discovery rates. DTASelect (10.1021/pr015504q) and swallow, an in-house developed software (v. 0.0.1, https://github.com/tzw-wen/kite), were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here all controlled FDRs were less than 1%. DTASelect-filter.txt files (output from DTASelect software) were converted to mzIdentML with dtaselect2mzid (https://github.com/proteomicsyates/DTASelect2MzId).

Project description:[NOTE: PF3D7_1470600 previously named RAP14 is now named RAP21]. HA-affinity purification - Purified late asexual parasites from parental and HA-tagged PF3D7_0105200 (RAP01-HA) and PF3D7_1470600 (RAP21-HA) lines were suspended in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 % Triton X-100, 1 mM AEBSF and EDTA-free protease inhibitor cocktail (Roche). After lysis by passing through a 26G needle 25 times, the soluble extracts were treated with 100 units of DNase I (NEB) for 10 min at room temperature and centrifuged at 14,000g for 15 min at 4C. The lysates were precleared with Dynabeads Protein A (Invitrogen) for 1h at 4C. The Rb anti-HA antibody (1:100, Abcam ab9110) was added in each sample and incubated overnight at 4C. Dynabeads Protein A were used to precipitate antibody-protein complexes and were washed with buffer A (1 % Triton X-100, 1 mM EDTA in PBS), buffer B (wash buffer A, 0.5 M NaCl) and buffer C (1 mM EDTA in PBS). Proteins were eluted using 0.1 M glycine, pH 2.8, and neutralized using 2 M Tris-HCl, pH 8.0. Then proteins were precipitated in 20% TCA followed by cold acetone washes. Multidimensional Protein Identification Technology - TCA-precipitated proteins were urea-denatured, reduced, alkylated, and digested with endoproteinase Lys-C (Roche) followed by modified trypsin (Promega). Peptide mixtures were loaded onto 100-um fused silica microcapillary columns packed with 5-um C18 reverse phase (Aqua, Phenomenex), strong cation exchange resin (Luna, Phenomenex), and 5-um C18 Aqua. Loaded microcapillary columns were placed in-line with a Quaternary Agilent 1100 series HPLC pump and a LTQ linear ion trap mass spectrometer equipped with a nano-LC electrospray ionization source (ThermoScientific, San Jose, CA). Fully automated 11-step MudPIT runs were carried out on the electrosprayed peptides. Tandem mass (MS/MS) spectra were interpreted using ProluCID against a database consisting of 5527 non-redundant (NR) Plasmodium falciparum 3D7 proteins (PlasmoDB-42 release), 36661 NR human proteins (NCBI, 2018-03-30 release), 419 usual contaminants (human keratins, IgGs, and proteolytic enzymes). To estimate false discovery rates (FDRs), the amino acid sequence of each NR protein entry was randomized, which resulted in a total search space of 85246 NR sequences. All cysteines were considered as fully carboxamidomethylated (+57 Da statically added), while methionine oxidation was searched as a differential modification. DTASelect and swallow, an in-house developed software (v.0.0.1, https://github.com/tzw-wen/kite), were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here, all controlled FDRs were less than 1.2%. Three immunoprecipitations were performed each for RAP01-HA and RAP21-HA along with 4 negative controls experiments.

Dataset Information

MudPIT analysis of the proteins associated with FLAG-Hoxa1 and FLAG-Hoxb1 purified from chromatin fractions isolated from mouse KH2ES

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets