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Combined proximity labeling and affinity purification−mass spectrometry workflow for mapping and visualizing protein interaction networks

Nature Protocols volume 15, pages 3182–3211 (2020)Cite this article

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Abstract

Affinity purification coupled with mass spectrometry (AP–MS) and proximity-dependent biotinylation identification (BioID) methods have made substantial contributions to interaction proteomics studies. Whereas AP−MS results in the identification of proteins that are in a stable complex, BioID labels and identifies proteins that are in close proximity to the bait, resulting in overlapping yet distinct protein identifications. Integration of AP–MS and BioID data has been shown to comprehensively characterize a protein’s molecular context, but interactome analysis using both methods in parallel is still labor and resource intense with respect to cell line generation and protein purification. Therefore, we developed the Multiple Approaches Combined (MAC)-tag workflow, which allows for both AP–MS and BioID analysis with a single construct and with almost identical protein purification and mass spectrometry (MS) identification procedures. We have applied the MAC-tag workflow to a selection of subcellular markers to provide a global view of the cellular protein interactome landscape. This localization database is accessible via our online platform (http://proteomics.fi) to predict the cellular localization of a protein of interest (POI) depending on its identified interactors. In this protocol, we present the detailed three-stage procedure for the MAC-tag workflow: (1) cell line generation for the MAC-tagged POI; (2) parallel AP–MS and BioID protein purification followed by MS analysis; and (3) protein interaction data analysis, data filtration and visualization with our localization visualization platform. The entire procedure can be completed within 25 d.

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Fig. 1: Schematic overview of the MAC-tag-based workflow.
Fig. 2: Verification of clones expressing a MAC-tagged POI.
Fig. 3: Schematic representation of the protein purification with Strep-Tactin beads for AP–MS and BioID (Steps 52−60).
Fig. 4: Schematic representation of the data analysis and visualization workflow.
Fig. 5: Application of the MS microscopy platform to an example MAC-tagged POI.

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Data availability

All the relevant files and testing examples can be downloaded from our website (http://proteomics.fi/). Other data that support this study are available from the corresponding author upon reasonable request.

Code availability

MS-microscopy Python code as well as R code for Shiny server are freely available on GitHub at https://github.com/kamms/ms-microscopy.

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Acknowledgements

We thank S. Miettinen for technical assistance and T. Öhman and E. Niemelä for critical reading and comments on the manuscript. We thank D. A. Yohannes for technical support of the MAC-tag online platform. Imaging was performed at the Light Microscopy Unit, Institute of Biotechnology. This study was supported by grants from the Academy of Finland (nos. 288475 and 294173), the Sigrid Jusélius Foundation, the Finnish Cancer Foundation, the University of Helsinki Three-year Research Grant, Biocentrum Helsinki, Biocentrum Finland, HiLIFE and the Instrumentarium Research Foundation.

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Authors and Affiliations

  1. Institute of Biotechnology, HiLIFE Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland

    Xiaonan Liu, Kari Salokas, Rigbe G. Weldatsadik, Lisa Gawriyski & Markku Varjosalo

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  1. Xiaonan Liu
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  2. Kari Salokas
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Contributions

M.V. and X.L. conceived the study and designed experiments. M.V., X.L., K.S., R.W. and G.L. performed experiments and data analysis. M.V., X.L., K.S., R.W. and G.L. participated in manuscript preparation. M.V. and X.L. wrote the manuscript.

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Correspondence to Markku Varjosalo.

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Key references using this protocol

Liu, X. et al. Nat. Commun. 9, 1188 (2018): https://www.nature.com/articles/s41467-018-03523-2

Kondelin, J. et al. EMBO Mol. Med. 10, e8552 (2018): https://www.embopress.org/doi/full/10.15252/emmm.201708552

Paakkola, T. et al. Hum. Mol. Genet. 27, 4288–4302 (2018): https://academic.oup.com/hmg/article/27/24/4288/5102903

Yellapragada, V. et al. Front. Endocrinol. 10, 48 (2019): https://www.frontiersin.org/articles/10.3389/fendo.2019.00048/full

Keskitalo, S. et al. Front. Immunol. 10, 2770 (2019): https://www.frontiersin.org/articles/10.3389/fimmu.2019.02770/full.

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Liu, X., Salokas, K., Weldatsadik, R.G. et al. Combined proximity labeling and affinity purification−mass spectrometry workflow for mapping and visualizing protein interaction networks. Nat Protoc 15, 3182–3211 (2020). https://doi.org/10.1038/s41596-020-0365-x

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