title: Proteomics-based identification of low-abundance signaling and regulatory protein complexes in native plant tissues.
abstract: Owing to the low abundance of signaling proteins and transcription factors, their protein complexes are not easily identified by classical proteomics. The isolation of these protein complexes from endogenous plant tissues (rather than plant cell cultures) is therefore an important technical challenge. Here, we describe a sensitive, quantitative proteomics-based procedure to determine the composition of plant protein complexes. The method makes use of fluorophore-tagged protein immunoprecipitation (IP) and label-free mass spectrometry (MS)-based quantification to correct for nonspecifically precipitated proteins. We provide procedures for the isolation of membrane-bound receptor complexes and transcriptional regulators from nuclei. The protocol consists of an IP step (~6 h) and sample preparation for liquid chromatography-tandem MS (LC-MS/MS; 2 d). We also provide a guide for data analysis. Our single-step affinity purification protocol is a good alternative to two-step tandem affinity purification (TAP), as it is shorter and relatively easy to perform. The data analysis by label-free quantification (LFQ) requires a cheaper and less challenging experimental setup compared with known labeling techniques in plants.
authors.affiliation: Plant Research International, Business Unit Bioscience, Wageningen, The Netherlands.
authors.forename: Cezary
authors.initials: C
authors.lastname: Smaczniak
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.forename: Sjef
authors.initials: S
authors.lastname: Boeren
authors.forename: Twan
authors.initials: T
authors.lastname: America
authors.forename: Walter
authors.initials: W
authors.lastname: van Dongen
authors.forename: Soenita S
authors.initials: SS
authors.lastname: Goerdayal
authors.forename: Sacco
authors.initials: S
authors.lastname: de Vries
authors.forename: Gerco C
authors.initials: GC
authors.lastname: Angenent
authors.forename: Kerstin
authors.initials: K
authors.lastname: Kaufmann
mesh_terms: D002853:Chromatography, Liquid; D005456:Fluorescent Dyes; D047468:Immunoprecipitation / Q000379:methods*; D047908:Intracellular Signaling Peptides and Proteins / Q000302:isolation & purification* / Q000378:metabolism; D046912:Multiprotein Complexes / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D046912:Multiprotein Complexes; D014157:Transcription Factors