title: An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes.
abstract: 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.
authors.affiliation: 1] Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie (VIB), Ghent, Belgium. [2] Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: Jelle
authors.initials: J
authors.lastname: Van Leene
authors.affiliation: 1] Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie (VIB), Ghent, Belgium. [2] Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: Dominique
authors.initials: D
authors.lastname: Eeckhout
authors.affiliation: 1] Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie (VIB), Ghent, Belgium. [2] Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: Bernard
authors.initials: B
authors.lastname: Cannoot
authors.affiliation: 1] Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie (VIB), Ghent, Belgium. [2] Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: Nancy
authors.initials: N
authors.lastname: De Winne
authors.affiliation: 1] Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie (VIB), Ghent, Belgium. [2] Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: Geert
authors.initials: G
authors.lastname: Persiau
authors.affiliation: 1] Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie (VIB), Ghent, Belgium. [2] Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: Eveline
authors.initials: E
authors.lastname: Van De Slijke
authors.affiliation: 1] Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie (VIB), Ghent, Belgium. [2] Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: Leen
authors.initials: L
authors.lastname: Vercruysse
authors.affiliation: 1] Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie (VIB), Ghent, Belgium. [2] Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: Maarten
authors.initials: M
authors.lastname: Dedecker
authors.affiliation: 1] Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie (VIB), Ghent, Belgium. [2] Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: Aurine
authors.initials: A
authors.lastname: Verkest
authors.affiliation: 1] Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie (VIB), Ghent, Belgium. [2] Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: Klaas
authors.identifier: 0000000347902725
authors.initials: K
authors.lastname: Vandepoele
authors.affiliation: 1] Department of Medical Protein Research, VIB, Ghent, Belgium. [2] Department of Biochemistry, Ghent University, Ghent, Belgium.
authors.forename: Lennart
authors.initials: L
authors.lastname: Martens
authors.affiliation: Centre for Proteome Analysis &Mass Spectrometry (CeProMa), University of Antwerp, Antwerp, Belgium.
authors.forename: Erwin
authors.initials: E
authors.lastname: Witters
authors.affiliation: 1] Department of Medical Protein Research, VIB, Ghent, Belgium. [2] Department of Biochemistry, Ghent University, Ghent, Belgium.
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
authors.affiliation: 1] Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie (VIB), Ghent, Belgium. [2] Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: Geert
authors.initials: G
authors.lastname: De Jaeger
mesh_terms: D000345:Affinity Labels; D017360:Arabidopsis / Q000737:chemistry* / Q000166:cytology* / Q000254:growth & development; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D007074:Immunoglobulin G; D046912:Multiprotein Complexes / Q000032:analysis / Q000302:isolation & purification*; D060066:Protein Interaction Maps; D036226:Seedlings / Q000166:cytology / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000345:Affinity Labels; D002352:Carrier Proteins; D007074:Immunoglobulin G; D046912:Multiprotein Complexes