Skip to main content
SpringerLink
Log in

Analyzing Protein Interactions by MAC-Tag Approaches

  • Protocol
  • First Online:
Protein-Protein Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2690))

Abstract

Proteomics methods such as affinity purification (AP) and proximity-dependent labeling (PL) coupled with mass spectrometry (MS) are currently commonly utilized to define interaction landscapes. BioID is one of the PL approaches, and it employs the expression of bait proteins fused to a nonspecific biotin ligase (BirA*), to induce in vivo biotinylation of proximal proteins. We developed the multiple approaches combined (MAC)-tag workflow, which allows for both AP and BioID analysis with a single construct and with almost identical protein purification and MS identification procedures. MAC-tag is a well-established method and has been widely used. Recent developed PL tags such as BioID2 and UltraID are smaller versions of BirA* with faster labeling efficiency. We therefore incorporate these tags into our system to develop MAC2-tag (containing BioID2) and MAC3-tag (containing UltraID) to overcome potential limitations of the original MAC-tag system and broaden the spectrum of applications for MAC-tags. Here, we describe a detailed procedure for the MAC-tag system workflow including cell line generation for the MAC/MAC2/MAC3-tagged protein of interest (POI), sample preparation for AP and PL protein purification, and MS analysis.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Gingras AC, Gstaiger M, Raught B et al (2007) Analysis of protein complexes using mass spectrometry. Nat Rev Mol Cell Biol 8(8):645–654. https://doi.org/10.1038/nrm2208

    Article  CAS  PubMed  Google Scholar 

  2. Varjosalo M, Sacco R, Stukalov A et al (2013) Interlaboratory reproducibility of large-scale human protein-complex analysis by standardized AP-MS. Nat Methods 10(4):307–314. https://doi.org/10.1038/nmeth.2400

    Article  CAS  PubMed  Google Scholar 

  3. Hein Marco Y, Hubner Nina C, Poser I et al (2015) A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell 163(3):712–723. https://doi.org/10.1016/j.cell.2015.09.053

    Article  CAS  PubMed  Google Scholar 

  4. Bonetta L (2010) Protein-protein interactions: interactome under construction. Nature 468(7325):851–854. https://doi.org/10.1038/468851a

    Article  CAS  PubMed  Google Scholar 

  5. Roux KJ, Kim DI, Raida M et al (2012) A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol 196(6):801–810. https://doi.org/10.1083/jcb.201112098

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kim DI, Jensen SC, Noble KA et al (2016) An improved smaller biotin ligase for. BioID Proximity Label 27(8):1188–1196. https://doi.org/10.1091/mbc.E15-12-0844

    Article  CAS  Google Scholar 

  7. Liu X, Huuskonen S, Laitinen T et al (2021) SARS-CoV-2-host proteome interactions for antiviral drug discovery. Mol Syst Biol 17(11):e10396. https://doi.org/10.15252/msb.202110396

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Liu X, Salokas K, Tamene F et al (2018) An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations. Nat Commun 9(1):1188. https://doi.org/10.1038/s41467-018-03523-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Salokas K, Liu X, Öhman T et al (2022) Physical and functional interactome atlas of human receptor tyrosine kinases. EMBO Report, p e54041. https://doi.org/10.15252/embr.202154041

    Book  Google Scholar 

  10. Göös H, Kinnunen M, Salokas K et al (2022) Human transcription factor protein interaction networks. Nat Commun 13(1):766. https://doi.org/10.1038/s41467-022-28341-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Chojnowski A, Sobota RM, Ong PF et al (2018) 2C-BioID: an advanced two component BioID system for precision mapping of protein interactomes. iScience 10:40–52. https://doi.org/10.1016/j.isci.2018.11.023

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Trinkle-Mulcahy L (2019) Recent advances in proximity-based labeling methods for interactome mapping. F1000Research 8. https://doi.org/10.12688/f1000research.16903.1

    Book  Google Scholar 

  13. Branon TC, Bosch JA, Sanchez AD et al (2018) Efficient proximity labeling in living cells and organisms with TurboID. Nat Biotechnol 36(9):880–887. https://doi.org/10.1038/nbt.4201

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhao X, Bitsch S, Kubitz L et al (2021) ultraID: a compact and efficient enzyme for proximity-dependent biotinylation in living cells. J bioRxiv. 2021.2006.2016.448656. https://doi.org/10.1101/2021.06.16.448656

  15. Varjosalo M, Keskitalo S, Van Drogen A et al (2013) The protein interaction landscape of the human CMGC kinase group. Cell Rep 3(4):1306–1320. https://doi.org/10.1016/j.celrep.2013.03.027

    Article  CAS  PubMed  Google Scholar 

  16. Wee P, Wang Z (2017) Epidermal growth factor receptor cell proliferation signaling pathways. Cancers (Basel) 9(5):52. https://doi.org/10.3390/cancers9050052

    Article  CAS  PubMed  Google Scholar 

  17. Vecchi M, Rudolph-Owen LA, Brown CL et al (1998) Tyrosine phosphorylation and proteolysis. Pervanadate-induced, metalloprotease-dependent cleavage of the ErbB-4 receptor and amphiregulin. J Biol Chem 273(32):20589–20595. https://doi.org/10.1074/jbc.273.32.20589

    Article  CAS  PubMed  Google Scholar 

  18. Bennett PA, Dixon RJ, Kellie S (1993) The phosphotyrosine phosphatase inhibitor vanadyl hydroperoxide induces morphological alterations, cytoskeletal rearrangements and increased adhesiveness in rat neutrophil leucocytes. J Cell Sci 106(Pt 3):891–901. https://doi.org/10.1242/jcs.106.3.891

    Article  CAS  PubMed  Google Scholar 

  19. Hietamäki J, Gregory LC, Ayoub S et al (2020) Loss-of-function variants in TBC1D32 underlie syndromic hypopituitarism. J Clin Endocrinol Metabol 105(6):1748–1758. https://doi.org/10.1210/clinem/dgaa078

    Article  Google Scholar 

  20. Yellapragada V, Liu X, Lund C et al (2019) MKRN3 interacts with several proteins implicated in puberty timing but does not influence GNRH1 expression. 10. https://doi.org/10.3389/fendo.2019.00048

  21. Liu X, Salokas K, Weldatsadik RG et al (2020) Combined proximity labeling and affinity purification−mass spectrometry workflow for mapping and visualizing protein interaction networks. Nat Protoc 15(10):3182–3211. https://doi.org/10.1038/s41596-020-0365-x

    Article  CAS  PubMed  Google Scholar 

  22. Meier F, Brunner A-D, Frank M et al (2020) diaPASEF: parallel accumulation–serial fragmentation combined with data-independent acquisition. Nat Methods 17(12):1229–1236. https://doi.org/10.1038/s41592-020-00998-0

    Article  CAS  PubMed  Google Scholar 

  23. Skowronek P, Meier F (2022) High-throughput mass spectrometry-based proteomics with dia-PASEF. Methodmol Biol (Clifton, NJ) 2456:15–27. https://doi.org/10.1007/978-1-0716-2124-0_2

    Article  Google Scholar 

  24. Orsburn BC (2021) Proteome discoverer – a community enhanced data processing suite for protein informatics. Proteomes 9(1):15. https://doi.org/10.3390/proteomes9010015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Tyanova S, Temu T, Cox J (2016) The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11(12):2301–2319. https://doi.org/10.1038/nprot.2016.136

    Article  CAS  PubMed  Google Scholar 

  26. Kong AT, Leprevost FV, Avtonomov DM et al (2017) MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry–based proteomics. Nat Methods 14(5):513–520. https://doi.org/10.1038/nmeth.4256

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Choi H, Larsen B, Lin ZY et al (2011) SAINT: probabilistic scoring of affinity purification-mass spectrometry data. Nat Methods 8(1):70–73. https://doi.org/10.1038/nmeth.1541

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank all members of the Varjosalo laboratory (https://www2.helsinki.fi/en/researchgroups/molecular-systems-biology), especially Tanja Turunen and Antti Tuhkala for optimization of the protocol. This work is funded by grants from the Academy of Finland (nos. 288475 and 294173), the Sigrid Jusélius Foundation, the Finnish Cancer Foundation, Biocentrum Finland, HiLIFE, and POLS (Norway Grants, no. 2020/37 / K / NZ4 / 02761).

Author information

Author notes

  1. Authors Xiaonan Liu and Kari Salokas have equally contributed to this chapter.

Authors and Affiliations

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

    Xiaonan Liu, Kari Salokas, Salla Keskitalo & Markku Varjosalo

  2. Department of Physiology, Faculty of Medical Sciences in Katowice, Medical University of Silesia, Katowice, Poland

    Xiaonan Liu

  3. Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Oviedo, Spain

    Patricia Martínez-Botía

Authors
  1. Xiaonan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kari Salokas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Salla Keskitalo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Patricia Martínez-Botía
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Markku Varjosalo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Markku Varjosalo .

Editor information

Editors and Affiliations

  1. Department of Biology, University of Alabama at Birmingham, Birmingham, AL, USA

    Shahid Mukhtar

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Liu, X., Salokas, K., Keskitalo, S., Martínez-Botía, P., Varjosalo, M. (2023). Analyzing Protein Interactions by MAC-Tag Approaches. In: Mukhtar, S. (eds) Protein-Protein Interactions. Methods in Molecular Biology, vol 2690. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3327-4_24

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3327-4_24

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3326-7

  • Online ISBN: 978-1-0716-3327-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation