Abstract
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.
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References
Ferro E, Trabalzini L (2013) The yeast two-hybrid and related methods as powerful tools to study plant cell signalling. Plant Mol Biol 83:287–301
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 109:303–308
Bücherl CA, Bader A, Westphal AH, Laptenok SP, Borst JW (2014) FRET-FLIM applications in plant systems. Protoplasma 251:383–394
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 24:426–437
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 139:1391–1398
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 21:1711–1722
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 7:2144–2158
Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906
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 26:1367–1372
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 10:1794–1805
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Wendrich, J.R., Boeren, S., Möller, B.K., Weijers, D., De Rybel, B. (2017). In Vivo Identification of Plant Protein Complexes Using IP-MS/MS. In: Kleine-Vehn, J., Sauer, M. (eds) Plant Hormones. Methods in Molecular Biology, vol 1497. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6469-7_14
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DOI: https://doi.org/10.1007/978-1-4939-6469-7_14
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6467-3
Online ISBN: 978-1-4939-6469-7
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