Chromatin is a very large sub-cellular structure likely containing thousands of proteins. Its analysis is complicated by the fact that chromatin is not defined by a sharp physical boundary such as a membrane. Chromatin is furthermore highly dynamic and changing during the cell cycle or changing environment.
As an important first step we are developing novel experimental concepts to isolate chromatin and to distinguish chromatin from contaminating proteins.
The Protein Composition of Mitotic Chromosomes Determined Using Multiclassifier Combinatorial Proteomics.
Shinya Ohta, Jimi-Carlo Bukowski-Wills, Luis Sanchez-Pulido, Flavia de Lima Alves, Laura Wood, Zhuo A. Chen, Melpi Platani, Lutz Fischer, Damien F. Hudson, Chris P. Ponting, Tatsuo Fukagawa, William C. Earnshaw, Juri Rappsilber. Cell. 2010 Sep 3;142(5):810-821. [Abstract] [PDF]
Some cellular substructures or organelles cannot be purified perfectly for proteomic analysis of their constituent proteins. Here we show that using machine learning tools to combine proteomics with other quantitative data can effectively add a final purification step in silico that separates genuine components from “hitchhikers”. Our approach sorted with 88% success the ~4,000 proteins identified by us in isolated mitotic chromosomes. The approach was validated by cloning 50 novel proteins, which yielded 13 novel centromere-associated proteins. Combining genetics with proteomics allowed also to study protein complexes without having to solubilize them from their native context in large subcellular structures.
Developmental Cell: Preview by Weijie Lan and Don W. Cleveland
Nature Methods: Research Highlight by Nicole Rusk – [PDF]
Faculty of 1000: Evaluation by Iain Cheeseman (Rating: Exceptional) and Silke Hauf (Rating: Exceptional)