Abstract
Identification of protein-protein interactions can be a critical step in understanding the function and regulation of a particular protein and for exploring intracellular signaling cascades. Affinity purification coupled to mass spectrometry (APMS) is a powerful method for isolating and characterizing protein complexes. This approach involves the tagging and subsequent enrichment of a protein of interest along with any stably associated proteins that bind to it, followed by the identification of the interacting proteins using mass spectrometry. The protocol described here offers a quick and simple method for routine sample preparation for APMS analysis of suitably tagged human cell lines.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Butland G, PeregrÃn-Alvarez JM, Li J et al (2005) Interaction network containing conserved and essential protein complexes in Escherichia coli. Nature 433:531–537. doi:10.1038/nature03239
Zhao R, Davey M, Hsu Y-C et al (2005) Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone. Cell 120:715–727. doi:10.1016/j.cell.2004.12.024
Gavin A-C, Aloy P, Grandi P et al (2006) Proteome survey reveals modularity of the yeast cell machinery. Nature 440:631–636. doi:10.1038/nature04532
Krogan NJ, Cagney G, Yu H et al (2006) Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440:637–643. doi:10.1038/nature04670
Sardiu ME, Cai Y, Jin J et al (2008) Probabilistic assembly of human protein interaction networks from label-free quantitative proteomics. Proc Natl Acad Sci U S A 105:1454–1459. doi:10.1073/pnas.0706983105
Sowa ME, Bennett EJ, Gygi SP, Harper JW (2009) Defining the human deubiquitinating enzyme interaction landscape. Cell 138:389–403. doi:10.1016/j.cell.2009.04.042
Behrends C, Sowa ME, Gygi SP, Harper JW (2010) Network organization of the human autophagy system. Nature 466:68–76. doi:10.1038/nature09204
Varjosalo M, Sacco R, Stukalov A et al (2013) Interlaboratory reproducibility of large-scale human protein-complex analysis by standardized AP-MS. Nat Methods 10:307–314. doi:10.1038/nmeth.2400
Roncagalli R, Hauri S, Fiore F et al (2014) Quantitative proteomics analysis of signalosome dynamics in primary T cells identifies the surface receptor CD6 as a Lat adaptor-independent TCR signaling hub. Nat Immunol 15:384–392. doi:10.1038/ni.2843
Guruharsha KG, Rual J-F, Zhai B et al (2011) A protein complex network of Drosophila melanogaster. Cell 147:690–703. doi:10.1016/j.cell.2011.08.047
Choi H, Larsen B, Lin Z-Y et al (2011) SAINT: probabilistic scoring of affinity purification-mass spectrometry data. Nat Methods 8:70–73. doi:10.1038/nmeth.1541
Teo G, Liu G, Zhang J et al (2014) SAINTexpress: improvements and additional features in Significance Analysis of INTeractome software. J Proteomics 100:37–43. doi:10.1016/j.jprot.2013.10.023
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Kwan, J.H.M., Emili, A. (2016). Simple and Effective Affinity Purification Procedures for Mass Spectrometry-Based Identification of Protein-Protein Interactions in Cell Signaling Pathways. In: Reinders, J. (eds) Proteomics in Systems Biology. Methods in Molecular Biology, vol 1394. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3341-9_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3341-9_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3339-6
Online ISBN: 978-1-4939-3341-9
eBook Packages: Springer Protocols