Proteomics-based identification of low-abundance signaling and regulatory protein complexes in native plant tissues

• Published in: Nature protocols Vol. 7; no. 12; pp. 2144 - 2158
• Main Authors: Smaczniak, Cezary, Li, Na, Boeren, Sjef, America, Twan, van Dongen, Walter, Goerdayal, Soenita S, de Vries, Sacco, Angenent, Gerco C, Kaufmann, Kerstin
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.12.2012; Nature Publishing Group
• Subjects: 631/1647/2067; 631/1647/527/296; 631/1647/664/2089; 631/449; Amino acids; Analytical Chemistry; Anatomy; Antibodies; arabidopsis-thaliana; Artificial chromosomes; bac transgeneomics; Biological Techniques; Botany; Cell culture; cells; Chromatography, Liquid; Computational Biology/Bioinformatics; Data analysis; Fluorescent Dyes; gene-expression; Immunoprecipitation - methods; Indigenous plants; Intracellular Signaling Peptides and Proteins - isolation & purification; Intracellular Signaling Peptides and Proteins - metabolism; Life Sciences; Liquid chromatography; Mass spectrometry; Methods; Microarrays; Multiprotein Complexes - isolation & purification; Multiprotein Complexes - metabolism; Observations; Organic Chemistry; Peptides; Plant proteins; Plant tissues; Proteins; Proteomics; Proteomics - methods; Protocol; quantification; quantitative proteomics; reveals; Sample preparation; Scientific imaging; strategy; Tandem Mass Spectrometry; Transcription Factors - isolation & purification; Transcription Factors - metabolism; Yeast; Netherlands; China
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2012.129

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.