A Quantitative Proteomics Tool To Identify DNA-Protein Interactions in Primary Cells or Blood
A Quantitative Proteomics Tool To Identify DNA-Protein Interactions in Primary Cells or Blood
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DOI：
10.1021/pr5009515
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发表时间：
2015-02-01
影响因子：
4.4
通讯作者：
Stunnenberg, Hendrik G.
中科院分区：
文献类型：
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作者：
Hubner, Nina C.;Nguyen, Luan N.;Stunnenberg, Hendrik G.
转录因子和基因组DNA之间的相互作用，特别是它们对疾病和细胞命运的影响，已经在全球范围内使用基于下一代测序的技术进行了广泛的研究。然而，这些方法不允许对与某些DNA序列结合的蛋白质复合物进行无偏见的研究。最近引入了结合定量质谱的来自粗裂解物的DNA下拉来缩小这一差距。然而，已建立的方案仅限于细胞系，因为它们基于代谢标记或需要大量材料。我们介绍了一种高通量兼容的DNA下拉，结合珠上消化与直接二甲基标记或无标记蛋白质定量。我们证明，我们的方法可以有效地识别转录因子结合到他们的共识DNA基序提取物从原代包皮成纤维细胞和外周血单核细胞（PBMC）新鲜分离的人供体。K562细胞和PBMC中近7000种蛋白质的绝对定量的核蛋白质组清楚地将差异相互作用与蛋白质丰度的差异联系起来，因此强调了选择相关细胞提取物用于任何相关相互作用的重要性。如rs6904029（一种与慢性淋巴细胞白血病高度相关的SNP）所示，我们的方法可以提供宝贵的功能数据，例如，通过与GWAS整合。
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.