A Quantitative Proteomics
Tool To Identify DNA–Protein
Interactions in Primary Cells or Blood
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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