Summary
Defining protein complexes is a vital aspect of cell biology because cellular processes are often carried out by stable protein complexes and their characterization often provides insights into their function. Accurate identification of the interacting proteins in macromolecular complexes is easiest after purification to near homogeneity. To this end, the tandem affinity purification (TAP) system with subsequent protein identification by high-throughput mass spectrometry was developed (1, 2) to systematically characterize native protein complexes and transient protein interactions under near-physiological conditions. The TAP tag containing two adjacent affinity purification tags (calmodulin-binding peptide and Staphylococcus aureus protein A) separated by a tobacco etch virus (TEV) protease cleavage site is fused with the open reading frame of interest. Using homologous recombination, a fusion library was constructed for the yeast Saccharomyces cerevisiae (3) in which the carboxy-terminal end of each predicted open reading frame is individually tagged in the chromosome so that the resulting fusion proteins are expressed under the control of their natural promoters (3). In this chapter, an optimized protocol for systematic protein purification and subsequent mass spectrometry-based protein identification is described in detail for the protein complexes of S. cerevisiae (4–6).
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Acknowledgments
The authors wish to thank Xinghua Guo and Gouqing Zhong for purifying the S. cerevisiae bait proteins and Shamanta Chandran, Peter Wong, and Constantine Christopoulos for assisting with mass spectrometry. This research was supported by grants from Genome Canada and the Ontario Genomics Institute to J.F.G. and A.E.
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Babu, M., Krogan, N.J., Awrey, D.E., Emili, A., Greenblatt, J.F. (2009). Systematic Characterization of the Protein Interaction Network and Protein Complexes in Saccharomyces cerevisiae Using Tandem Affinity Purification and Mass Spectrometry. In: Stagljar, I. (eds) Yeast Functional Genomics and Proteomics. Methods in Molecular Biology, vol 548. Humana Press. https://doi.org/10.1007/978-1-59745-540-4_11
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DOI: https://doi.org/10.1007/978-1-59745-540-4_11
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