Plant Hormones pp 147-158 | Cite as

In Vivo Identification of Plant Protein Complexes Using IP-MS/MS

  • Jos R. Wendrich
  • Sjef Boeren
  • Barbara K. Möller
  • Dolf WeijersEmail author
  • Bert De Rybel
Part of the Methods in Molecular Biology book series (MIMB, volume 1497)


Individual proteins often function as part of a protein complex. The identification of interacting proteins is therefore vital to understand the biological role and function of the studied protein. Here we describe a method for the in vivo identification of nuclear, cytoplasmic, and membrane-associated protein complexes from plant tissues using a strategy of immunoprecipitation followed by tandem mass spectrometry. By performing quantitative mass spectrometry measurements on biological triplicates, relative abundance of proteins in GFP-tagged complexes compared to background controls can be statistically evaluated to identify high-confidence interactors. We detail the entire workflow of this approach.

Key words

Protein–protein interaction Arabidopsis Immunoprecipitation Tandem mass spectrometry Protein complex identification GFP 


  1. 1.
    Ferro E, Trabalzini L (2013) The yeast two-hybrid and related methods as powerful tools to study plant cell signalling. Plant Mol Biol 83:287–301CrossRefPubMedGoogle Scholar
  2. 2.
    Albrecht C, Boutrot F, Segonzac C, Schwessinger B, Gimenez-Ibanez S, Chinchilla D, Rathjen JP, de Vries SC, Zipfel C (2012) Brassinosteroids inhibit pathogen-associated molecular pattern-triggered immune signaling independent of the receptor kinase BAK1. Proc Natl Acad Sci U S A 109:303–308CrossRefPubMedGoogle Scholar
  3. 3.
    Bücherl CA, Bader A, Westphal AH, Laptenok SP, Borst JW (2014) FRET-FLIM applications in plant systems. Protoplasma 251:383–394CrossRefPubMedGoogle Scholar
  4. 4.
    De Rybel B, Möller B, Yoshida S, Grabowicz I, Barbier de Reuille P, Boeren S, Smith RS, Borst JW, Weijers D (2013) A bHLH complex controls embryonic vascular tissue establishment and indeterminate growth in Arabidopsis. Dev Cell 24:426–437CrossRefPubMedGoogle Scholar
  5. 5.
    Saiga S, Möller B, Watanabe-Taneda A, Abe M, Weijers D, Komeda Y (2012) Control of embryonic meristem initiation in Arabidopsis by PHD-finger protein complexes. Development 139:1391–1398CrossRefPubMedGoogle Scholar
  6. 6.
    Zwiewka M, Feraru E, Möller B, Hwang I, Feraru MI, Kleine-Vehn J, Weijers D, Friml J (2011) The AP-3 adaptor complex is required for vacuolar function in Arabidopsis. Cell Res 21:1711–1722CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Smaczniak C, Li N, Boeren S, America T, van Dongen W, Goerdayal SS, de Vries S, Angenent GC, Kaufmann K (2012) Proteomics-based identification of low-abundance signaling and regulatory protein complexes in native plant tissues. Nat Protoc 7:2144–2158CrossRefPubMedGoogle Scholar
  8. 8.
    Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906CrossRefPubMedGoogle Scholar
  9. 9.
    Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372CrossRefPubMedGoogle Scholar
  10. 10.
    Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10:1794–1805CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Jos R. Wendrich
    • 1
  • Sjef Boeren
    • 1
  • Barbara K. Möller
    • 1
    • 4
  • Dolf Weijers
    • 1
    Email author
  • Bert De Rybel
    • 1
    • 2
    • 3
  1. 1.Laboratory of BiochemistryWageningen UniversityWageningenThe Netherlands
  2. 2.Department of Plant Systems Biology, Flemish Institute of BiotechnologyVIB Ghent UniversityGhentBelgium
  3. 3.Department of Plant Biotechnology and BioinformaticsGhent UniversityGhentBelgium
  4. 4.Department of Plant Systems BiologyVIB Ghent UniversityGhentBelgium

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