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Mapping Protein–Protein Interactions Using Affinity Purification and Mass Spectrometry

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Plant Genomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1610))

Abstract

The mapping of protein–protein interaction (PPI) networks and their dynamics are crucial steps to deciphering the function of a protein and its role in cellular pathways, making it critical to have comprehensive knowledge of a protein’s interactome. Advances in affinity purification and mass spectrometry technology (AP-MS) have provided a powerful and unbiased method to capture higher-order protein complexes and decipher dynamic PPIs. However, the unbiased calling of nonspecific interactions and the ability to detect transient interactions remains challenging when using AP-MS, thereby hampering the detection of biologically meaningful complexes. Additionally, there are plant-specific challenges with AP-MS, such as a lack of protein-specific antibodies, which must be overcome to successfully identify PPIs. Here we discuss and describe a protocol designed to bypass the traditional challenges of AP-MS and provide a roadmap to identify bona fide PPIs in plants.

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Acknowledgments

D. A. N. provided the pB7-HFN binary vector used for generating transgenic lines expressing 3XFLAG-6XHis-tagged bait proteins. The trypsin digestion, sample preparation for mass spectrometry, and MASCOT analysis were technically supported by Dr. Jean Kanyo at the W. M. Keck Biotechnology Resource Laboratory at Yale University. We also want to thank Dr. Shirin Bahmanyar, Dr. Man-Wah Li, Dr. Wei Liu, and Olivia Compagna for their useful discussion and comments. This research is supported by NSF (IOS-1456796 to D. A. N. and IOS-1548538 to J. M. G. and DGE-1122492 to AMF), NIH (T32 GM007499 to A.F.), the Gruber Foundataion (A.F.), Yale University Forest B. H. and Elizabeth D. W. Brown Fund Endowed Postdoctoral Fellowship (C.L.) and the Rudolph J. Anderson Postdoctoral Fellowship (C.L.).

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Authors and Affiliations

  1. Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, 06511, USA

    Chin-Mei Lee, Christopher Adamchek, Ann Feke & Joshua M. Gendron

  2. Donald Danforth Plant Science Center, St. Louis, MO, 63132, USA

    Dmitri A. Nusinow

Authors
  1. Chin-Mei Lee
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  2. Christopher Adamchek
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  3. Ann Feke
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  4. Dmitri A. Nusinow
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  5. Joshua M. Gendron
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Corresponding author

Correspondence to Joshua M. Gendron .

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Editors and Affiliations

  1. Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Vienna, Austria

    Wolfgang Busch

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Lee, CM., Adamchek, C., Feke, A., Nusinow, D.A., Gendron, J.M. (2017). Mapping Protein–Protein Interactions Using Affinity Purification and Mass Spectrometry. In: Busch, W. (eds) Plant Genomics. Methods in Molecular Biology, vol 1610. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7003-2_15

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  • DOI: https://doi.org/10.1007/978-1-4939-7003-2_15

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7001-8

  • Online ISBN: 978-1-4939-7003-2

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