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A method to resolve the composition of heterogeneous affinity-purified protein complexes assembled around a common protein by chemical cross-linking, gel electrophoresis and mass spectrometry

Nature Protocols volume 8, pages 75–97 (2013)Cite this article

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Abstract

Protein complexes form, dissociate and re-form in order to perform specific cellular functions. In this two-pronged protocol, noncovalent protein complexes are initially isolated by affinity purification for subsequent identification of the components by liquid chromatography high-resolution mass spectrometry (LC-MS) on a hybrid LTQ Orbitrap Velos. In the second prong of the approach, the affinity-purification strategy includes a chemical cross-linking step to 'freeze' a series of concurrently formed, heterogeneous protein subcomplex species that are visualized by gel electrophoresis. This branch of the methodology amalgamates standard and well-practiced laboratory methods to reveal compositional changes that occur in protein complex architecture. By using mouse N-terminally tagged streptavidin-binding peptide–hemagglutinin–TANK-binding kinase 1 (SH-TBK1), we chemically cross-linked the affinity-purified complex of SH-TBK1 with the homobifunctional lysine-specific reagent bis(sulfosuccinimidyl) suberate (BS3), and we separated the resultant protein complexes by denaturation and by silver-stained one- and two-dimensional SDS-PAGE. We observed a range of cross-linked TBK1 complexes of variable pI and Mr and confirmed them by immunoblotting. LC-MS analysis of in situ–digested cross-linked proteins shows differences in the composition of the TBK1 subcomplexes. The protocol is inherently simple and can be readily extended to the investigation of a range of protein complexes. From cell lysis to data generation by LC-MS, the protocol takes approximately 2.5 to 5.5 d to perform.

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Figure 1: Schematic overview of the entire protocol.
Figure 2: SH-tagged TBK1 protein complexes separated by 1D (lanes 1–3) and 2D (regions 4 and 5) SDS-PAGE) on 3–8% (wt/vol) Tris-acetate gradient gels.

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Acknowledgements

We thank T. Bürckstümmer from Haplogen GmbH, Vienna, for providing the mouse SH-TBK1 entry vector, A.C. Müller for valuable advice on iTRAQ quantification and desalting techniques; and all the members of the Superti-Furga and Bennett laboratories for helpful discussions. Work in our laboratory is supported by the Austrian Academy of Sciences, the Austrian Federal Ministry for Science and Research (Gen-Au projects, APP-III and BIN-III), the Austrian Science Fund FWF and the Central Bank of the Republic of Austria. E.L.R. and R.S. are supported by the GenAu APP-III program (no. 820965), and M.L.H. by the Central Bank of the Republic of Austria (no. 14252).

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Author notes

  1. Elena L Rudashevskaya and Roberto Sacco: These authors contributed equally to this work.

Authors and Affiliations

  1. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

    Elena L Rudashevskaya, Roberto Sacco, Marie L Huber, Giulio Superti-Furga & Keiryn L Bennett

  2. Department of Paediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria

    Klaus Kratochwill

  3. Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases, Medical University of Vienna, Vienna, Austria

    Marie L Huber

  4. Institute of Molecular Systems Biology, ETH, Zurich, Switzerland

    Matthias Gstaiger

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  1. Elena L Rudashevskaya
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Contributions

E.L.R. developed the protocol, conducted the experiments, interpreted the data and drafted the manuscript. R.S. developed sections of the protocol, participated in optimizing the tandem affinity purification methodology, wrote sections of the manuscript and provided valuable feedback in the review process. K.K. provided intellectual and technical expertise in optimization of the 2D SDS-PAGE, performed the 2D SDS-PAGE experiments, interpreted the gel images and wrote the 2D SDS-PAGE sections of the manuscript. M.L.H. provided technical support with 1D SDS-PAGE and sample preparation for LC-MS, and assisted in writing sections of the manuscript and proofreading of the revised versions. M.G. and G.S.-F. provided intellectual expertise on tandem affinity purifications and valuable feedback during the revision process. K.L.B. and E.L.R. conceived the notion of the protocol. K.L.B. was responsible for project supervision, data interpretation, manuscript writing and providing grant support.

Corresponding author

Correspondence to Keiryn L Bennett.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

SH-tagged TBK1 protein complexes. Complexes are separated by: (A) one- and (B) two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (1D- and 2D-SDS-PAGE) on 3-8% tris-acetate gradient gels. The different TBK1 sub-complexes and interacting proteins are: (a) SH-TBK1_M+TBK1_H, HS90A, HS90B, AZI2, TANK; (b) SH-TBK1_M+TBK1_H, TANK; (c) SH-TBK1_M+TBK1_H, HS90A, HS90B; (d) HS90A, HS90B; (e) HS90A, HS90B, TBKBP1; (f) SH-TBK1_M, TBK1_H, HS90A, HS90B; (g) SH-TBK1_M, TBK1_H, TBKBP1; (h) SH-TBK1_M, TBK1_H. Bold numbers in (A) are the molecular mass ranges of the 1D-SDS-PAGE. Unique peptide counts, spectral counts (or number of MSMS spectra matched to peptides), and sequence coverage are given for each sub-complex. (PDF 333 kb)

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Rudashevskaya, E., Sacco, R., Kratochwill, K. et al. A method to resolve the composition of heterogeneous affinity-purified protein complexes assembled around a common protein by chemical cross-linking, gel electrophoresis and mass spectrometry. Nat Protoc 8, 75–97 (2013). https://doi.org/10.1038/nprot.2012.133

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