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

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


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 



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).


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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

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