Identification of Brassinosteroid Signaling Complexes by Coimmunoprecipitation and Mass Spectrometry
A combination of coimmunoprecipitation (coIP) of tagged proteins followed by protein identification and quantitation using Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LCMS/MS) has proven to be a reliable method to qualitatively characterize membrane-bound receptor complexes from plants. Success depends on a range of parameters, such as abundance and stability of the complex and functionality of the tagged receptors, efficiency of the protein complex isolation procedure, MS equipment, and analysis software in use. In this Chapter, we focus on the use of one of the green fluorescent protein-tagged receptors of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, of which SERK3, also known as BRASSINOSTEROID INSENSITIVE1 (BRI1) ASSOCIATED KINASE1 (BAK1), is a coreceptor of BRI1. Like BRI1 itself, SERK3 is a leucine-rich repeat receptor kinase (LRR RK) with a single-pass transmembrane domain. The latest updated laboratory protocol is presented as well as examples of data analysis and typical results obtained. Potential drawbacks of the procedure employed for plant membrane proteins will be pointed out.
Key wordsProtein–protein interaction Arabidopsis Immunoprecipitation Tandem mass spectrometry Protein complex identification GFP
We thank Jos Wendrich (Biochemistry, Wageningen University, Netherlands) and Bert de Rybel (VIB-Ghent University, Belgium) for providing the updated immunoprecipitation protocols currently in use.
- 6.Wendrich JR, Boeren S, Möller BK et al (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, 3rd edn. Humana Press, Totowa, NJ 1497:147–158Google Scholar