Protein Complexes Characterization in Arabidopsis thaliana by Tandem Affinity Purification Coupled to Mass Spectrometry Analysis

  • Jean Bigeard
  • Delphine Pflieger
  • Jean Colcombet
  • Loïc Gérard
  • Hakim Mireau
  • Heribert HirtEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1171)


Proteins are major elements participating in all the key functions of the cells. They rarely fulfill their physiological roles in an autonomous way but rather act as part of more complex cellular machines. Indeed they can bind different types of molecules (proteins, nucleic acids, metabolites, etc.), via stable or transient interactions, depending on their nature and functions. The identification of the molecular partners of a given protein is hence essential to better understand its roles, regulation, and mechanisms of action.

This chapter describes the use of a tandem affinity purification approach followed by mass spectrometry analysis to try to identify and characterize the proteins involved in protein complexes in Arabidopsis thaliana and decipher some mechanisms of regulation of the modules. Important elements to consider in such an approach are first extensively exposed in the introduction. This technique, in combination with complementary approaches like yeast two-hybrid and bimolecular fluorescence complementation, can be an interesting source of data to identify and characterize in vivo protein complexes.

Key words

Arabidopsis thaliana Protein complexes Tandem affinity purification Mass spectrometry Posttranslational modifications 



This work was partly funded by the French plant genomics program, Génoplante.


  1. 1.
    Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Seraphin B (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17:1030–1032PubMedCrossRefGoogle Scholar
  2. 2.
    Gavin AC, Bosche M, Krause R, Grandi P, Marzioch M, Bauer A, Schultz J, Rick JM, Michon AM, Cruciat CM et al (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415:141–147PubMedCrossRefGoogle Scholar
  3. 3.
    Krogan NJ, Cagney G, Yu H, Zhong G, Guo X, Ignatchenko A, Li J, Pu S, Datta N, Tikuisis AP et al (2006) Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440:637–643PubMedCrossRefGoogle Scholar
  4. 4.
    Gully D, Moinier D, Loiseau L, Bouveret E (2003) New partners of acyl carrier protein detected in Escherichia coli by tandem affinity purification. FEBS Lett 548:90–96PubMedCrossRefGoogle Scholar
  5. 5.
    Knuesel M, Wan Y, Xiao Z, Holinger E, Lowe N, Wang W, Liu X (2003) Identification of novel protein–protein interactions using a versatile mammalian tandem affinity purification expression system. Mol Cell Proteomics 2:1225–1233PubMedCrossRefGoogle Scholar
  6. 6.
    Forler D, Kocher T, Rode M, Gentzel M, Izaurralde E, Wilm M (2003) An efficient protein complex purification method for functional proteomics in higher eukaryotes. Nat Biotechnol 21:89–92PubMedCrossRefGoogle Scholar
  7. 7.
    Rohila JS, Chen M, Cerny R, Fromm ME (2004) Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants. Plant J 38:172–181PubMedCrossRefGoogle Scholar
  8. 8.
    Li Y (2010) Commonly used tag combinations for tandem affinity purification. Biotechnol Appl Biochem 55:73–83PubMedCrossRefGoogle Scholar
  9. 9.
    Schmidt TG, Koepke J, Frank R, Skerra A (1996) Molecular interaction between the Strep-tag affinity peptide and its cognate target, streptavidin. J Mol Biol 255:753–766PubMedCrossRefGoogle Scholar
  10. 10.
    Bornhorst JA, Falke JJ (2000) Purification of proteins using polyhistidine affinity tags. Methods Enzymol 326:245–254PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Rossignol M (2006) Proteomic analysis of phosphorylated proteins. Curr Opin Plant Biol 9:538–543PubMedCrossRefGoogle Scholar
  12. 12.
    Kersten B, Agrawal GK, Durek P, Neigenfind J, Schulze W, Walther D, Rakwal R (2009) Plant phosphoproteomics: an update. Proteomics 9:964–988PubMedCrossRefGoogle Scholar
  13. 13.
    Russell WK, Park ZY, Russell DH (2001) Proteolysis in mixed organic-aqueous solvent systems: applications for peptide mass mapping using mass spectrometry. Anal Chem 73:2682–2685PubMedCrossRefGoogle Scholar
  14. 14.
    Olsen JV, de Godoy LM, Li G, Macek B, Mortensen P, Pesch R, Makarov A, Lange O, Horning S, Mann M (2005) Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4:2010–2021PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Jean Bigeard
    • 1
  • Delphine Pflieger
    • 2
  • Jean Colcombet
    • 1
  • Loïc Gérard
    • 1
  • Hakim Mireau
    • 3
  • Heribert Hirt
    • 1
    Email author
  1. 1.URGV Plant GenomicsUMR INRA-1165/CNRS-ERL8196/Université d’Evry Val d’EssonneEvry CedexFrance
  2. 2.Laboratoire Analyse et Modélisation pour la Biologie et l’EnvironnementCNRS UMR 8587, Université d’Evry Val d’EssonneEvry CedexFrance
  3. 3.Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, INRA Centre de Versailles-GrignonVersailles CedexFrance

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