Over the past few years, we have been using this technology to identify interaction partners for the developmental regulators we are studying. We hereby provide a detailed protocol and several examples of publication in which this technology was successfully applied.
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. Over the past few years, we have been using IP-MS/MS technology for the in vivo identification of nuclear, cytoplasmic, and membrane-associated protein complexes from plant tissues. 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.
A detailed protocol can be found following this link or is available upon request.
Andrés-Colás N, Zhu Q, Takenaka M, De Rybel B, Weijers D, Van Der Straeten D. (2017). Multiple PPR protein interactions are involved in the RNA editing system in Arabidopsis mitochondria and plastids. Proc Natl Acad Sci U S A. 2017 Aug 15;114(33):8883-8888. PMID: 28761003
Muñoz A, Mangano S, González-García MP, Contreras R, Sauer M, De Rybel B, Weijers D, Sánchez-Serrano JJ, Sanmartín M, Rojo E. (2017). RIMA-Dependent Nuclear Accumulation of IYO Triggers Auxin-Irreversible Cell Differentiation in Arabidopsis. Plant Cell. 2017 Mar;29(3):575-588. PMID: 28223441
Wendrich JR, Boeren S, Möller BK, Weijers D, De Rybel B. (2017) In Vivo Identification of Plant Protein Complexes Using IP-MS/MS. Methods in Molecular Biology. 2017;1497:147-158 PMID: 27864765
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. Developmental Cell. 2013 Feb 25;24(4):426-37. PMID: 23415953