Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada.
Mol Syst Biol. 2010 Dec 21;6:448. doi: 10.1038/msb.2010.104.
We previously reported a novel affinity purification (AP) method termed modified chromatin immunopurification (mChIP), which permits selective enrichment of DNA-bound proteins along with their associated protein network. In this study, we report a large-scale study of the protein network of 102 chromatin-related proteins from budding yeast that were analyzed by mChIP coupled to mass spectrometry. This effort resulted in the detection of 2966 high confidence protein associations with 724 distinct preys. mChIP resulted in significantly improved interaction coverage as compared with classical AP methodology for ∼75% of the baits tested. Furthermore, mChIP successfully identified novel binding partners for many lower abundance transcription factors that previously failed using conventional AP methodologies. mChIP was also used to perform targeted studies, particularly of Asf1 and its associated proteins, to allow for a understanding of the physical interplay between Asf1 and two other histone chaperones, Rtt106 and the HIR complex, to be gained.
我们之前报道了一种新的亲和纯化（AP）方法，称为改良染色质免疫沉淀（mChIP），它允许与它们相关的蛋白质网络一起选择性地富集与 DNA 结合的蛋白质。在这项研究中，我们报告了一项针对酿酒酵母 102 种染色质相关蛋白的蛋白质网络的大规模研究，该研究通过 mChIP 与质谱法相结合进行分析。这项工作检测到 2966 种与 724 种不同猎物具有高置信度的蛋白质关联。与经典的 AP 方法相比，mChIP 显著提高了约 75%的诱饵的相互作用覆盖率。此外，mChIP 还成功地为许多以前使用传统 AP 方法无法检测到的低丰度转录因子鉴定了新的结合伙伴。mChIP 还用于进行靶向研究，特别是对 Asf1 及其相关蛋白的研究，以了解 Asf1 与另外两种组蛋白伴侣 Rtt106 和 HIR 复合物之间的物理相互作用。
