Dataset Information

MudPIT analysis of proteins extracted from mouse KH2ES cell nuclei and enriched by binding to immobilized DNA fragments

ABSTRACT: 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.

INSTRUMENT(S): LTQ

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

SUBMITTER: Laurence Florens

PROVIDER: MSV000086596 | MassIVE | Mon Dec 14 15:06:00 GMT 2020

SECONDARY ACCESSION(S): PXD023129

REPOSITORIES: MassIVE

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Publications

Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression.

Singh Narendra Pratap NP De Kumar Bony B Paulson Ariel A Parrish Mark E ME Scott Carrie C Zhang Ying Y Florens Laurence L Krumlauf Robb R

Journal of developmental biology 20210203 1

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-oc ...[more]

PMID: 33546292

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Project description: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.

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