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Methods Mol Biol. 2009;492:283-93. doi: 10.1007/978-1-59745-493-3_17.

Chemical cross-linking for protein-protein interaction studies.

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Department of Chemistry, Washington State University, Pullman, WA, USA.


Most proteins function through protein complex assemblies. Defining and mapping protein complex networks are crucial elements in the fundamental understanding of biological processes. The ability to measure protein-protein interactions in biological systems has undergone significant advances in the past decade due to emergence and growth of numerous new molecular biology and mass spectrometry technologies. Chemical cross-linking, along with yeast two-hybrid, fluorescence resonant energy transfer (FRET), and co-immunoprecipitation have become important tools for detection and characterization of protein-protein interactions. Individual protein members in a noncovalent complex assembly remain in close proximity which is within the reach of the two reactive groups of a cross-linker. Thus cross-linking reactions have potential for linking two interacting proteins which exist in close proximity. In general, chemical cross-linking experiments are carried out by first linking the interacting proteins through covalent bonds followed by a series of well-established protocols -- SDS-PAGE, in-gel digestion, and shotgun LC/MS/MS for identification of the cross-linked proteins. These approaches have been employed for both mapping topology of protein complex in vitro and determining the protein interaction partners in vivo.

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