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Cell Rep. 2015 Oct 20;13(3):621-633. doi: 10.1016/j.celrep.2015.09.009. Epub 2015 Oct 8.

A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in Human Cells.

Author information

1
Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Laval University, Quebec City, QC G1V 4G2, Canada; St-Patrick Research Group in Basic Oncology and Laval University Cancer Research Center, Quebec City, QC G1R 3S3, Canada.
2
Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Laval University, Quebec City, QC G1V 4G2, Canada.
3
Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Laval University, Quebec City, QC G1V 4G2, Canada. Electronic address: yannick.doyon@crchudequebec.ulaval.ca.

Abstract

Conventional affinity purification followed by mass spectrometry (AP-MS) analysis is a broadly applicable method used to decipher molecular interaction networks and infer protein function. However, it is sensitive to perturbations induced by ectopically overexpressed target proteins and does not reflect multilevel physiological regulation in response to diverse stimuli. Here, we developed an interface between genome editing and proteomics to isolate native protein complexes produced from their natural genomic contexts. We used CRISPR/Cas9 and TAL effector nucleases (TALENs) to tag endogenous genes and purified several DNA repair and chromatin-modifying holoenzymes to near homogeneity. We uncovered subunits and interactions among well-characterized complexes and report the isolation of MCM8/9, highlighting the efficiency and robustness of the approach. These methods improve and simplify both small- and large-scale explorations of protein interactions as well as the study of biochemical activities and structure-function relationships.

PMID:
26456817
DOI:
10.1016/j.celrep.2015.09.009
[Indexed for MEDLINE]
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