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.

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Figure 1: Workflow of the TAP platform for Arabidopsis cell cultures and seedlings.
Figure 2: Schematic representation of the GSrhino tag fused to either the N or C terminus of the bait protein of interest.
Figure 3: SDS-PAGE analysis of TAP eluates, showing the high specificity and reproducibility of the GS-TAP protocol.
Figure 4: Overview of the different multisite Gateway cloning strategies for fusion of the GSrhino tag to the bait protein.
Figure 5: Schemes for the determination of nonspecific proteins and transcript expression breadth of the nonspecific gene list.

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Acknowledgements

J.V.L. is a postdoctoral Fellow of the Research Foundation-Flanders. We thank S. Ghorbani for practical support; A. Staes and J. Vandenbussche for support with mass spectrometry; G. Gonnelli for help with data analysis; K. Verleye, J.V. Driessche and N. Helderwert for general support; and A. Bleys for help in preparing the manuscript.

Author information

J.V.L., D.E. and G.D.J. designed the research. B.C., N.D.W., G.P., E.V.D.S., L.V. and J.V.L. performed experiments. D.E., J.V.L., K.V., L.M. and G.D.J. analyzed the data. K.G. and E.W. provided protocols for LC-MS/MS analysis. M.D., A.V., K.V., K.G., L.M. and E.W. commented on the manuscript. J.V.L., D.E. and G.D.J. wrote the manuscript.

Correspondence to Geert De Jaeger.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Distribution of the average Normalized Spectral Abundance Factors (NSAF) of non-specific proteins.

Non-specific proteins were grouped in subsets by the number of different baitgroups the non-specific proteins were identified in.

Supplementary Figure 2 Identification of two non-specific proteins as genuine interactions.

Identification of two Actin-related proteins as genuine interactions using AN3 as bait, either with TAP on cell culture or on seedlings. In all TAP experiments where ARP4 or ARP7 were identified, the difference was calculated between the NSAF in that sample and the minimal NSAF value of 0.4% for low abundant non-specific proteins (See Supplementary Fig. 1).

Supplementary Figure 3 Setup of the assembled IgG Sepharose Polyprep column during binding of the total protein extract onto the IgG beads.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Table 1 (PDF 703 kb)

Supplementary Table 2

Excel list of 760 nonspecific proteins, based on the occurrence of a co-purified protein among 543 TAP experiments using 115 different bait proteins, classified into 62 unrelated bait groups. In column three, the number of baitgroups in which a protein was identified, is shown. (XLS 110 kb)

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Van Leene, J., Eeckhout, D., Cannoot, B. et al. An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes. Nat Protoc 10, 169–187 (2015). https://doi.org/10.1038/nprot.2014.199

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