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

Jelle Van Leene UGent, Dominique Eeckhout UGent, Bernard Cannoot UGent, Nancy De Winne UGent, Geert Persiau UGent, Eveline Van De Slijke UGent, Leen Vercruysse, Maarten Dedecker, Aurine Verkest, Klaas Vandepoele UGent, et al. (2015) NATURE PROTOCOLS. 10(1). p.169-187
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 (similar to 7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; similar to 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.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
FLORAL DIP, TRANSCRIPTION, SACCHAROMYCES-CEREVISIAE, LEAF DEVELOPMENT, DIVISION, PROTEOMICS, THALIANA, AGROBACTERIUM-MEDIATED TRANSFORMATION, SUSPENSION-CULTURES, ANAPHASE-PROMOTING COMPLEX/CYCLOSOME
journal title
NATURE PROTOCOLS
Nat. Protoc.
volume
10
issue
1
pages
169 - 187
Web of Science type
Article
Web of Science id
000347159400011
JCR category
BIOCHEMICAL RESEARCH METHODS
JCR impact factor
9.646 (2015)
JCR rank
2/77 (2015)
JCR quartile
1 (2015)
ISSN
1754-2189
DOI
10.1038/nprot.2014.199
project
Bioinformatics: from nucleotids to networks (N2N)
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
5835945
handle
http://hdl.handle.net/1854/LU-5835945
date created
2015-02-02 16:05:51
date last changed
2016-12-19 15:42:55
@article{5835945,
  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 (similar to 7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; similar to 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.},
  author       = {Van Leene, Jelle and Eeckhout, Dominique and Cannoot, Bernard and De Winne, Nancy and Persiau, Geert and Van De Slijke, Eveline and Vercruysse, Leen and Dedecker, Maarten and Verkest, Aurine and Vandepoele, Klaas and Martens, Lennart and Witters, Erwin and Gevaert, Kris and De Jaeger, Geert},
  issn         = {1754-2189},
  journal      = {NATURE PROTOCOLS},
  keyword      = {FLORAL DIP,TRANSCRIPTION,SACCHAROMYCES-CEREVISIAE,LEAF DEVELOPMENT,DIVISION,PROTEOMICS,THALIANA,AGROBACTERIUM-MEDIATED TRANSFORMATION,SUSPENSION-CULTURES,ANAPHASE-PROMOTING COMPLEX/CYCLOSOME},
  language     = {eng},
  number       = {1},
  pages        = {169--187},
  title        = {An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes},
  url          = {http://dx.doi.org/10.1038/nprot.2014.199},
  volume       = {10},
  year         = {2015},
}

Chicago
Van Leene, Jelle, Dominique Eeckhout, Bernard Cannoot, Nancy De Winne, Geert Persiau, Eveline Van De Slijke, Leen Vercruysse, et al. 2015. “An Improved Toolbox to Unravel the Plant Cellular Machinery by Tandem Affinity Purification of Arabidopsis Protein Complexes.” Nature Protocols 10 (1): 169–187.
APA
Van Leene, J., Eeckhout, D., Cannoot, B., De Winne, N., Persiau, G., Van De Slijke, E., Vercruysse, L., et al. (2015). An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes. NATURE PROTOCOLS, 10(1), 169–187.
Vancouver
1.
Van Leene J, Eeckhout D, Cannoot B, De Winne N, Persiau G, Van De Slijke E, et al. An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes. NATURE PROTOCOLS. 2015;10(1):169–87.
MLA
Van Leene, Jelle, Dominique Eeckhout, Bernard Cannoot, et al. “An Improved Toolbox to Unravel the Plant Cellular Machinery by Tandem Affinity Purification of Arabidopsis Protein Complexes.” NATURE PROTOCOLS 10.1 (2015): 169–187. Print.