PXD000782

PXD000782 is an original dataset announced via ProteomeXchange.

Title	A quantitative proteomics tool to identify DNA-protein interactions in primary cells or blood
Description	The interaction between transcription factors and genomic DNA forms the basis for spatio-temporal control of gene expression. Therefore, these interactions and their impact on disease and cellular fate are extensively studied on a global level, mainly using techniques based on next-generation sequencing. These techniques, however, do not allow an unbiased study of proteins or entire protein complexes that bind to a certain DNA sequence. In recent years, DNA pull-downs followed by quantitative mass spectrometry were introduced to close this gap. Established protocols, however, are based on metabolic labeling techniques or require enormous amounts of cellular material, thus restricting the method to cell lines grown in culture. Furthermore, they require substantial amount of expertise, thus keeping this technique restricted to a limited number of laboratories. Here, we introduce a high-throughput compatible, LC-MS/MS based DNA pull-down that combines on-bead digestion with direct dimethyl labeling or label-free protein quantification. We demonstrate, that our method can efficiently identify transcription factors binding to their known consensus DNA motifs when using nuclear extracts from model cell lines. Subsequently, we apply the method to study DNA-protein interactions in primary foreskin fibroblasts and peripheral blood mononuclear cells (PBMCs) freshly isolated from human donors. We show that the same DNA sequence binds different sets of proteins in an established model cell line as opposed to PBMCs. This stresses the importance of selecting relevant cell extracts for any interaction in question. In-depth nuclear proteomes with absolute quantification of close to 7,000 proteins in K562 cells and PBMCs clearly link these differential interactions to differences in protein abundance. In conclusion, our approach, applicable to primary material and capable of profiling DNA-protein interactions in high-throughput, will likely prove itself as a useful screening platform and will provide invaluable functional data, for example through integration with large scale GWAS.
HostingRepository	PRIDE
AnnounceDate	2015-02-05
AnnouncementXML	Submission_2015-02-05_00:30:40.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Nina Hubner
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	iodoacetamide derivatized residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2014-02-28 03:16:04	ID requested
1	2015-02-04 07:37:38	announced
2	2015-02-04 08:04:30	announced	Another attempt at submitting PX XML, due to web service error.
⏵ 3	2015-02-05 00:30:41	announced	Updated project metadata.

Hubner NC, Nguyen LN, Hornig NC, Stunnenberg HG, A quantitative proteomics tool to identify DNA-protein interactions in primary cells or blood. J Proteome Res, 14(2):1315-29(2015) [pubmed]

curator keyword: Biomedical, Technical

submitter keyword: DNA pull-down, blood, primary cells, PBMCs

Hendrik G. Stunnenberg
contact affiliation	Radboud University Nijmegen, The Netherlands
contact email	h.stunnenberg@ncmls.ru.nl
lab head
Nina Hubner
contact affiliation	Radboud University Nijmegen, The Netherlands
contact email	n.hubner@ncmls.ru.nl
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/01/PXD000782

PRIDE project URI

• PRIDE
  1. PXD000782
     1. Label: PRIDE project
     2. Name: A quantitative proteomics tool to identify DNA-protein interactions in primary cells or blood