PXD023129

PXD023129 is an original dataset announced via ProteomeXchange.

Title	MudPIT analysis of proteins extracted from mouse KH2ES cell nuclei and enriched by binding to immobilized DNA fragments
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.
HostingRepository	MassIVE
AnnounceDate	2021-01-28
AnnouncementXML	Submission_2021-01-28_12:31:50.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Laurence Florens
SpeciesList	scientific name: Mus musculus; common name: house mouse; NCBI TaxID: 10090;
ModificationList	S-carboxamidomethyl-L-cysteine; L-methionine sulfoxide
Instrument	LTQ

Revision	Datetime	Status	ChangeLog Entry
0	2020-12-14 15:06:32	ID requested
⏵ 1	2021-01-28 12:31:50	announced

no publication

submitter keyword: KH2 embryonic stem cells, Hoxb1, DNA binding

Laurence Florens
contact affiliation	The Stowers Institute for Medical Research
contact email	laf@stowers.org
lab head
Laurence Florens
contact affiliation	Stowers Institute for Medical Research
contact email	laf@stowers.org
dataset submitter

MassIVE dataset URI

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