Abstract
Protein complexes and protein–protein interactions (PPIs) are fundamental for most biological functions. Deciphering the extensive protein interaction networks that occur within cellular contexts has become a logical extension to the human genome project. Proteome-scale interactome analysis of mammalian systems requires efficient methods for accurately detecting PPIs with specific considerations for the intrinsic technical challenges of mammalian genome manipulation. In this chapter, we outline in detail an innovative lentiviral-based functional proteomic approach that can be used to rapidly characterize protein complexes from a broad range of mammalian cell lines. This method integrates the following key features: (1) lentiviral elements for efficient delivery of tagged constructs into mammalian cell lines; (2) site-specific Gateway™ recombination sites for easy cloning; (3) versatile epitope-tagging system for flexible affinity purification strategies; and (4) LC-MS-based protein identification using tandem mass spectrometry.
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Acknowledgments
We thank Anthony B. Mak and Denitza Roudeva for critical reading of the manuscript. This project is supported by grants from the Canadian Institutes of Health Research (CIHR) and Ontario Research Fund to JG and AE. ZN is supported by a CIHR fellowship.
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Ni, Z., Olsen, J.B., Emili, A., Greenblatt, J.F. (2011). Identification of Mammalian Protein Complexes by Lentiviral-Based Affinity Purification and Mass Spectrometry. In: Cagney, G., Emili, A. (eds) Network Biology. Methods in Molecular Biology, vol 781. Humana Press. https://doi.org/10.1007/978-1-61779-276-2_2
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DOI: https://doi.org/10.1007/978-1-61779-276-2_2
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