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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M800447-MCP200v1 8/4/870    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lambert, J.-P. Articles by Figeys, D. Search for Related Content PubMed PubMed Citation Articles by Lambert, J.-P. Articles by Figeys, D. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 8:870-882, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

A Novel Proteomics Approach for the Discovery of Chromatin-associated Protein Networks^*,S

Jean-Philippe Lambert, Leslie Mitchell, Adam Rudner, Kristin Baetz^¶ and Daniel Figeys^||

From the Ottawa Institute of Systems Biology and Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada

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.

^|| A Canada Research Chair in Proteomics and Systems Biology. To whom correspondence may be addressed. E-mail: dfigeys{at}uottawa.ca

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