Abstract
Protein-protein interaction mapping has progressed rapidly in recent years, enabling the completion of several high throughput studies. However, knowledge of physical interactions is limited for numerous classes of proteins, such as chromatin-bound proteins, because of their poor solubility when bound to DNA. To address this problem, we have developed a novel method, termed modified chromatin immunopurification (mChIP), that allows for the efficient purification of protein-DNA macromolecules, enabling subsequent protein identification by mass spectrometry. mChIP consists of a single affinity purification step whereby chromatin-bound protein networks are isolated from mildly sonicated and gently clarified cellular extracts using magnetic beads coated with antibodies. We applied the mChIP method in Saccharomyces cerevisiae cells expressing endogenously tandem affinity purification (TAP)-tagged histone H2A or the histone variant Htz1p and successfully co-purified numerous chromatin-bound protein networks as well as DNA. We further challenged the mChIP procedure by purifying three chromatin-bound bait proteins that have proven difficult to purify by traditional methods: Lge1p, Mcm5p, and Yta7p. The protein interaction networks of these three baits dramatically expanded our knowledge of their chromatin environments and illustrate that the innovative mChIP procedure enables an improved characterization of chromatin-associated proteins.
Highlights
Protein-protein interaction mapping has progressed rapidly in recent years, enabling the completion of several high throughput studies
MChIP Facilitates the Purification of Protein Networks Bound to DNA—The study of proteins associated with chromatin is difficult
We found that most of the proteins expected to be associated with chromatin were lost during the sample preparation, in the centrifugation steps
Summary
Protein-protein interaction mapping has progressed rapidly in recent years, enabling the completion of several high throughput studies. Knowledge of physical interactions is limited for numerous classes of proteins, such as chromatin-bound proteins, because of their poor solubility when bound to DNA To address this problem, we have developed a novel method, termed modified chromatin immunopurification (mChIP), that allows for the efficient purification of protein-DNA macromolecules, enabling subsequent protein identification by mass spectrometry. We and others have successfully performed large scale studies of protein-protein interactions in both yeast and human cells by immunopurification and high throughput mass spectrometry [5,6,7,8,9,10]. A well established protocol for the study of histones, utilizes the low solubility of the histone proteins that are complexed with DNA to precipitate them from the cellular matrix; histones are subsequently resolubilized for
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