Chapter 17
Context-Specific and Proximity-Dependent Labeling
for the Proteomic Analysis of Spatiotemporally Defined
Protein Complexes with Split-BioID
Cinthia Amaya Ramirez, Stefanie Egetemaier, and Julien Be ´ thune
Abstract
Proximity-dependent labeling techniques such as BioID and APEX2 allow the biotinylation of proteins
proximal to a protein of interest in living cells. Following streptavidin pulldown and mass spectrometry
analysis, this enables the identification of native protein–protein interactions. Here we describe split-BioID,
a protein-fragment complementation assay that increases the resolution of BioID. Using this technique,
context-specific protein complexes can be resolved.
Key words BioID, Protein-fragment complementation assay, Protein–protein interactions, Biotin,
Proteomics
1 Introduction
In all living organisms, thousands of proteins mediate most cellular functions. To do so, proteins usually do not act alone. Rather,
they assemble with other proteins to build dynamic macromolecular complexes that can remodel according to the exact functions
that need to be exerted. The protein–protein interactions (PPI)
involved in such complexes are often deregulated in disease and
are thus promising targets for therapeutics [1]. Identifying and
characterizing PPI networks are hence of prime importance when
trying to understand how a protein of interest (POI) works. To
complement classical pulldown approaches in which the POI is
isolated by affinity purification (AP) and co-purifying proteins
identified by mass spectrometry (MS) analysis, proximitydependent biotinylation techniques were recently introduced
that allow the labeling of proteins vicinal to the POI in living
cells. Two such techniques are currently available: APEX2- and
BioID-mediated labeling [2, 3]. The former relies on an engineered peroxidase that activates a biotin-phenol substrate that is
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_17, © Springer Science+Business Media, LLC, part of Springer Nature 2021
303
Context-Specific and Proximity-Dependent Labeling
for the Proteomic Analysis of Spatiotemporally Defined
Protein Complexes with Split-BioID
Cinthia Amaya Ramirez, Stefanie Egetemaier, and Julien Be ´ thune
Abstract
Proximity-dependent labeling techniques such as BioID and APEX2 allow the biotinylation of proteins
proximal to a protein of interest in living cells. Following streptavidin pulldown and mass spectrometry
analysis, this enables the identification of native protein–protein interactions. Here we describe split-BioID,
a protein-fragment complementation assay that increases the resolution of BioID. Using this technique,
context-specific protein complexes can be resolved.
Key words BioID, Protein-fragment complementation assay, Protein–protein interactions, Biotin,
Proteomics
1 Introduction
In all living organisms, thousands of proteins mediate most cellular functions. To do so, proteins usually do not act alone. Rather,
they assemble with other proteins to build dynamic macromolecular complexes that can remodel according to the exact functions
that need to be exerted. The protein–protein interactions (PPI)
involved in such complexes are often deregulated in disease and
are thus promising targets for therapeutics [1]. Identifying and
characterizing PPI networks are hence of prime importance when
trying to understand how a protein of interest (POI) works. To
complement classical pulldown approaches in which the POI is
isolated by affinity purification (AP) and co-purifying proteins
identified by mass spectrometry (MS) analysis, proximitydependent biotinylation techniques were recently introduced
that allow the labeling of proteins vicinal to the POI in living
cells. Two such techniques are currently available: APEX2- and
BioID-mediated labeling [2, 3]. The former relies on an engineered peroxidase that activates a biotin-phenol substrate that is
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_17, © Springer Science+Business Media, LLC, part of Springer Nature 2021
303