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J Proteome Res. 2015 Feb 6;14(2):1315-29. doi: 10.1021/pr5009515. Epub 2015 Jan 8.

A quantitative proteomics tool to identify DNA-protein interactions in primary cells or blood.

Author information

1
Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen , Nijmegen 6525 GA, The Netherlands.

Abstract

Interactions between transcription factors and genomic DNA, and in particular their impact on disease and cell fate, have been extensively studied on a global level using techniques based on next-generation sequencing. These approaches, however, do not allow an unbiased study of protein complexes that bind to certain DNA sequences. DNA pulldowns from crude lysates combined with quantitative mass spectrometry were recently introduced to close this gap. Established protocols, however, are restricted to cell lines because they are based on metabolic labeling or require large amounts of material. We introduce a high-throughput-compatible DNA pulldown 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 consensus DNA motifs in extracts from primary foreskin fibroblasts and peripheral blood mononuclear cells (PBMCs) freshly isolated from human donors. Nuclear proteomes with absolute quantification of nearly 7000 proteins in K562 cells and PBMCs clearly link differential interactions to differences in protein abundance, hence stressing the importance of selecting relevant cell extracts for any interaction in question. As shown for rs6904029, a SNP highly associated with chronic lymphocytic leukemia, our approach can provide invaluable functional data, for example, through integration with GWAS.

KEYWORDS:

DNA pulldown; DNA‚ąíprotein interactions; dimethyl labeling; protein complex; quantitative mass spectrometry; transcription factor

PMID:
25546135
DOI:
10.1021/pr5009515
[Indexed for MEDLINE]

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