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An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells

Nature Methods volume 3, pages 1013–1019 (2006)Cite this article

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Abstract

Tandem affinity purification (TAP) is a generic two-step affinity purification protocol that enables the isolation of protein complexes under close-to-physiological conditions for subsequent analysis by mass spectrometry. Although TAP was instrumental in elucidating the yeast cellular machinery, in mammalian cells the method suffers from a low overall yield. We designed several dual-affinity tags optimized for use in mammalian cells and compared the efficiency of each tag to the conventional TAP tag. A tag based on protein G and the streptavidin-binding peptide (GS-TAP) resulted in a tenfold increase in protein-complex yield and improved the specificity of the procedure. This allows purification of protein complexes that were hitherto not amenable to TAP and use of less starting material, leading to higher success rates and enabling systematic interaction proteomics projects. Using the well-characterized Ku70-Ku80 protein complex as an example, we identified both core elements as well as new candidate effectors.

*Note: In the version of this article initially published online, the GS-TAP tag in Figure 3b was incorrectly identified as a GC-TAP tag, and the email address for material requests (materials@cemm.oeaw.ac.at) was omitted from the methods section. The errors have been corrected for all versions of the article.

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Figure 1: Schematic representation of TAP.
Figure 2: Comparison of yTAP and AC-TAP.
Figure 3: Comparison of the different TAP tag variants.
Figure 4: Quantification of the overall process.
Figure 5: TAP of the Ku70-Ku80 complex.
Figure 6: Quantitative overview of the TAP procedure.

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Change history

  • 06 October 2006

    In the version of this article initially published online, the GS-TAP tag in Figure 3b was incorrectly identified as a GC-TAP tag, and the email address for material requests (materials@cemm.oeaw.ac.at) was omitted from the methods section. The errors have been corrected for all versions of the article.

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Acknowledgements

We thank M. Brehme for help with graphical illustrations. We also thank J.-M. Peters (Institute for Molecular Pathology, Vienna), S.P. Jackson (Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge), M. Mann (Max Planck Institute for Biochemistry, Martinsried) and R. Aebersold (Eidgenoessische Technische Hochschule, Zuerich) for critical reading of the manuscript. We thank M. Planyavsky for preparation of the TAP samples for analysis by mass spectrometry and the entire Superti-Furga laboratory for suggestions. T.B. is supported by a research fellowship (BU 2180/1-1) from the German Research Foundation (DFG). CeMM is supported by the Austrian Academy of Sciences. Work described here was funded by the Austrian Proteomics Platform-II of the GenAU Program of the Austrian Ministry of Education and Research, and by a grant from the Austrian National Bank.

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  1. Research Center for Molecular Medicine (CeMM), Lazarettgasse 19/3, Vienna, 1090, Austria

    Tilmann Bürckstümmer, Keiryn L Bennett, Adrijana Preradovic, Gregor Schütze, Oliver Hantschel, Giulio Superti-Furga & Angela Bauch

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  1. Tilmann Bürckstümmer
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Correspondence to Giulio Superti-Furga.

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Competing interests

G.S.-F. has received a consultancy salary from the drug discovery/proteomics company Cellzome Inc. as a member of its scientific advisory board.

Supplementary information

Supplementary Fig. 1

Detailed characterization of the AC- and the GS-TAP-tag. (PDF 539 kb)

Supplementary Fig. 2

GS-TAP-Ku70: Biochemical activity and specificity control. (PDF 2485 kb)

Supplementary Fig. 3

The GS-TAP tag allows for a single-step purification. (PDF 1526 kb)

Supplementary Table 1

Lists of interactors associated with Ku70 and nonspecific interactors as identified my mass spectrometry analysis. (DOC 2098 kb)

Supplementary Methods (DOC 44 kb)

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Bürckstümmer, T., Bennett, K., Preradovic, A. et al. An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells. Nat Methods 3, 1013–1019 (2006). https://doi.org/10.1038/nmeth968

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