Journal Article

An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes

Van Leene, Jelle, De Winne, Nancy, Vandepoele, Klaas, Witters, Erwin, Verkest, Aurine, Van De Slijke, Eveline, Vercruysse, Leen, Eeckhout, Dominique, Cannoot, Bernard, Martens, Lennart, Persiau, Geert, Dedecker, Maarten, De Jaeger, Geert, Gevaert, Kris

2015

Overview

A platform for isolating low-abundance protein complexes from Arabidopsis seedlings and cell cultures is described. Its power resides in an improved TAP tag combined with ultrasensitive MS and filtering against a list of nonspecific proteins. Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (∼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; ∼5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/1647/2230/2232 / 631/1647/296 / 631/1647/334/2244/710 / 631/337/475/2290 / Affinity / Affinity Labels / Analysis / Analytical Chemistry / Arabidopsis / Arabidopsis - chemistry / Arabidopsis - cytology / Arabidopsis - growth & development / Arabidopsis thaliana / Baits / Biological Techniques / Carrier Proteins - genetics / Carrier Proteins - metabolism / Cell culture / Chromatography, Liquid - methods / Computational Biology/Bioinformatics / Genetic aspects / Immunoglobulin G / Life Sciences / Liquid chromatography / Mass spectrometry / Mass spectroscopy / Microarrays / Multiprotein Complexes - analysis / Multiprotein Complexes - isolation & purification / Organic Chemistry / Physiological aspects / Proteases / Protein G / Protein interaction / Protein Interaction Maps / Protein purification / Protein-protein interactions / Proteins / protocol / Purification / Sample preparation / Seedlings / Seedlings - cytology / Seedlings - metabolism / Streptavidin / Tandem Mass Spectrometry - methods