title: Identification of Brassinosteroid Signaling Complexes by Coimmunoprecipitation and Mass Spectrometry.
abstract: 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.
authors.affiliation: Laboratory of Biochemistry, Wageningen UR, 6708, WE, Wageningen, The Netherlands.
authors.forename: Walter
authors.initials: W
authors.lastname: van Dongen
authors.affiliation: Laboratory of Biochemistry, Wageningen UR, 6708, WE, Wageningen, The Netherlands.
authors.forename: Luc
authors.initials: L
authors.lastname: van Heerde
authors.affiliation: Laboratory of Biochemistry, Wageningen UR, 6708, WE, Wageningen, The Netherlands.
authors.forename: Sjef
authors.initials: S
authors.lastname: Boeren
authors.affiliation: Laboratory of Biochemistry, Wageningen UR, 6708, WE, Wageningen, The Netherlands.
authors.forename: Sacco C
authors.initials: SC
authors.lastname: de Vries
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D060406:Brassinosteroids / Q000378:metabolism; D002853:Chromatography, Liquid; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation / Q000379:methods; D058977:Molecular Sequence Annotation; D030821:Plants, Genetically Modified; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D036226:Seedlings / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D029681:Arabidopsis Proteins; D060406:Brassinosteroids; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011494:Protein Kinases; C463660:BAK1 protein, Arabidopsis; C108270:BRI1 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases
keywords: Arabidopsis; GFP; Immunoprecipitation; Protein complex identification; Protein–protein interaction; Tandem mass spectrometry