Advertisement

Direct Identification of Biotinylated Proteins from Proximity Labeling (Spot-BioID)

  • Song-Yi Lee
  • Jeong Kon Seo
  • Hyun-Woo RheeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2008)

Abstract

Recently, proximity labeling has been developed to map spatially localized proteomes in live cells. Usually, these methods employ enzymatic biotinylation of the proximal proteins with reactive biotin species. The labeled proteins may contain biotinylated modifications, which can be enriched by streptavidin beads through affinity purification. However, during the bead enrichment process, unlabeled proteins can be enriched to have specific binding affinity toward the biotinylated proteins or high binding affinity to the bead surface. If the unlabeled proteins remain attached to the beads after washing and are analyzed by mass spectrometry (MS) using the conventional workflow for the unlabeled peptidome, they would appear as proximal proteins in the targeted space. However, the unlabeled proteins, including the specific interaction partners of the biotinylated proteins, are false positives for proximity labeling. Including the unlabeled proteome in the identification list for proximity labeling does not provide a clear picture of the local proteome in the targeted space. This chapter is a detailed protocol of the first direct identification method (Spot-BioID) for identifying biotin-labeled proteomes of promiscuous biotin ligase (pBirA) labeling.

Key words

Proximity labeling Promiscuous biotin ligase (pBirA) Engineered ascorbate peroxidase (APEX) Biotin-labeled peptide Mass spectrometry (MS) Spot-ID 

Notes

Acknowledgment

This work was supported by the National Research Foundation of Korea (NRF-2016R1C1B2013956, NRF-2018M3A9G4078528, NRF-2018K2A9A2A08000087, NRF-2019R1A2C3008463) and the Organelle Network Research Center (NRF-2017R1A5A1015366). S.Y.L. and H. W. R. thank general support from the KBRI basic research program through Korea Brain Research Institute funded by Ministry of Science and ICT (17-BR-01). H.W.R is supported by Research Resettlement Fund for the new faculty of Seoul National University. S.Y.L. is supported by the BK21 Plus (Division of Chemstry & Molecular Engineering, Seoul National University).

References

  1. 1.
    Kim DI et al (2014) Probing nuclear pore complex architecture with proximity-dependent biotinylation. Proc Natl Acad Sci U S A 111:E2453CrossRefGoogle Scholar
  2. 2.
    Bendayan M (2001) Worth its weight in gold. Science 291:1363CrossRefGoogle Scholar
  3. 3.
    Roux KJ et al (2012) A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol 196:801–810CrossRefGoogle Scholar
  4. 4.
    Lee SY et al (2016) Proximity-directed labeling reveals a new rapamycin-induced heterodimer of FKBP25 and FRB in live cells. ACS Cent Sci 2:506–516CrossRefGoogle Scholar
  5. 5.
    Firat-Karalar EN et al (2014) Proximity interactions among centrosome components identify regulators of centriole duplication. Curr Biol 24:664–670CrossRefGoogle Scholar
  6. 6.
    Lambert J-P et al (2015) Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes. J Proteome 118:81–94CrossRefGoogle Scholar
  7. 7.
    Rhee HW et al (2013) Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 339:1328–1331CrossRefGoogle Scholar
  8. 8.
    Hung V et al (2014) Proteomic mapping of the human mitochondrial intermembrane space in live cells via ratiometric APEX tagging. Mol Cell 55:332–341CrossRefGoogle Scholar
  9. 9.
    Kim DI et al (2018) BioSITe: a method for direct detection and quantitation of site-specific biotinylation. J Proteome Res 17:759–769CrossRefGoogle Scholar
  10. 10.
    Lee SY et al (2017) Architecture mapping of the inner mitochondrial membrane proteome by chemical tools in live cells. J Am Chem Soc 139:3651–3662CrossRefGoogle Scholar
  11. 11.
    Udeshi ND et al (2017) Antibodies to biotin enable large-scale detection of biotinylation sites on proteins. Nat Methods 14:1167CrossRefGoogle Scholar
  12. 12.
    Schiapparelli LM et al (2014) Direct detection of biotinylated proteins by mass spectrometry. J Proteome Res 13:3966–3978CrossRefGoogle Scholar
  13. 13.
    Uezu A et al (2016) Identification of an elaborate complex mediating postsynaptic inhibition. Science 353(6304):1123–1129CrossRefGoogle Scholar
  14. 14.
    Branon TC et al (2018) Efficient proximity labeling in living cells and organisms with turboid. Nature Biotechnology 36(9):880–887CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of ChemistrySeoul National UniversitySeoulRepublic of Korea
  2. 2.Department of ChemistryUlsan National Institute of Science and Technology (UNIST)UlsanRepublic of Korea
  3. 3.UNIST Central Research Facilities (UCRF)Ulsan National Institute of Science and Technology (UNIST)UlsanRepublic of Korea

Personalised recommendations