title: Identification of post-translational modifications of plant protein complexes.
abstract: Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved. Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs. This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.
authors.affiliation: School of Life Sciences, University of Warwick.
authors.forename: Sophie J M
authors.initials: SJ
authors.lastname: Piquerez
authors.affiliation: The Sainsbury Laboratory, Norwich Research Park.
authors.forename: Alexi L
authors.initials: AL
authors.lastname: Balmuth
authors.affiliation: The Sainsbury Laboratory, Norwich Research Park.
authors.forename: Jan
authors.initials: J
authors.lastname: Sklenář
authors.affiliation: School of Life Sciences, University of Warwick; The Sainsbury Laboratory, Norwich Research Park.
authors.forename: Alexandra M E
authors.initials: AM
authors.lastname: Jones
authors.affiliation: Research School of Biology, The Australian National University.
authors.forename: John P
authors.initials: JP
authors.lastname: Rathjen
authors.affiliation: School of Life Sciences, University of Warwick; V.Ntoukakis@warwick.ac.uk.
authors.forename: Vardis
authors.initials: V
authors.lastname: Ntoukakis
mesh_terms: D017360:Arabidopsis / Q000737:chemistry / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000302:isolation & purification / Q000378:metabolism; D002498:Centrifugation / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods*; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
chemical_list: D029681:Arabidopsis Proteins; D010940:Plant Proteins