Proteomics > Which service should I request? > Interaction Proteomics > Proximity Labeling
In proximity-dependent biotinylation experiments a protein of interest (bait) is fused with a promiscuous enzymatic activity that allows marking proteins in close vicinity. Typical enzymes are protein biotin ligases or peroxidases. Biotin ligases (BirA, BioID2, TurboID, miniTurboID) use biotin as a substrate and modify lysine residues in nearby proteins, whereas peroxidases (HRP, APEX, APEX2) convert biotin-phenol to short lived radicals in the presence of peroxide, finally leading to tyrosine biotinylation of proteins. This biotin modification is afterwards used as purification handle exploiting conventional streptavidin capture methods. In contrast to AP-MS workflows this allows using very harsh lysis and purification conditions, since prey proteins are not enriched via weak interactions. This also allows detecting rather transient kiss-and-run type of contacts between proteins, or marking the proteome in a cellular compartment.
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There is excellent reviews on proximity-dependent biotinylation including experimental design. We suggest reading the following:
Most important (as always in experimental sciences) are controls. The following paragraph summarises the most important aspects:
"Controls should thus minimally include conditions that mimic endogenous biotinylation (such as no enzyme fused to the bait protein, or untransfected cells) and conditions that reproduce promiscuous biotinylation (e.g. enzyme alone expressed throughout the cell, and/or fused to an irrelevant polypeptide such as the Green Fluorescent Protein). To properly model promiscuous background, the control polypeptide must also be expressed to at least the same level as the bait protein, and should at least partially occupy the same intracellular locale. In cases where a protein of interest localizes to a specific subcellular location, it may also be useful to include an enzyme fusion that is specifically localized to this structure."
On top one should apply the general principles of good experimental design (replication, blocking and randomisation) as outlined for the AP-MS workflow.
"Proximity-dependent biotinylation approaches performed on a single bait protein can generate hundreds to thousands of putative partner identifications (with parameters such as the amount of material and the sensitivity of the mass spectrometer significantly influencing these numbers). Importantly, however, in most cases the majority of these proteins are not true proximity partners for the bait of interest. Many are instead: biotin-labeled in the absence of the recombinant enzyme (e.g. endogenously biotinylated proteins such as mitochondrial carboxylases); proteins that are promiscuously biotinylated with most baits (e.g. with BioID in HEK293 cells, filamin A belongs to this category), or; proteins that bind non-specifically to the affinity support (sepharose or other bead types)."
Proximity Labeling
Table of contents
General description
In proximity-dependent biotinylation experiments a protein of interest (bait) is fused with a promiscuous enzymatic activity that allows marking proteins in close vicinity. Typical enzymes are protein biotin ligases or peroxidases. Biotin ligases (BirA, BioID2, TurboID, miniTurboID) use biotin as a substrate and modify lysine residues in nearby proteins, whereas peroxidases (HRP, APEX, APEX2) convert biotin-phenol to short lived radicals in the presence of peroxide, finally leading to tyrosine biotinylation of proteins. This biotin modification is afterwards used as purification handle exploiting conventional streptavidin capture methods. In contrast to AP-MS workflows this allows using very harsh lysis and purification conditions, since prey proteins are not enriched via weak interactions. This also allows detecting rather transient kiss-and-run type of contacts between proteins, or marking the proteome in a cellular compartment.
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Workflow
Our default workflow for the analysis of proximity-dependent biotinylation experiments is very similar to the workflow for AP-MS samples.1. Protein digestion
The interaction between streptavidin and biotin is the strongest known non-covalent interaction, hence we usually employ on-bead digestion to release capture prey proteins from the purification resin.2. LC-MS analysis
Check the Affinity Proteomics MS page for up to date information3. Data analysis
Also the data analysis pipeline is very similar to AP-MS. Captured proteins are quantified and enrichments are calculated in contrast to control samples where the enzymatic activity was expressed out of bait context.Experimental Design
There is excellent reviews on proximity-dependent biotinylation including experimental design. We suggest reading the following:
- Samavarchi-Tehrani, P., Samson, R. & Gingras, A.-C. Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches. Mol Cell Proteomics 19, 757–773 (2020). https://doi.org/10.1074/mcp.R120.001941
- Gingras, A.-C., Abe, K. T. & Raught, B. Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles. Curr Opin Chem Biol 48, 44–54 (2019). https://doi.org/10.1016/j.cbpa.2018.10.017
- Richards, A. L., Eckhardt, M. & Krogan, N. J. Mass spectrometry‐based protein–protein interaction networks for the study of human diseases. Mol Syst Biol 17, e8792 (2021). https://doi.org/10.15252/msb.20188792
Most important (as always in experimental sciences) are controls. The following paragraph summarises the most important aspects:
"Controls should thus minimally include conditions that mimic endogenous biotinylation (such as no enzyme fused to the bait protein, or untransfected cells) and conditions that reproduce promiscuous biotinylation (e.g. enzyme alone expressed throughout the cell, and/or fused to an irrelevant polypeptide such as the Green Fluorescent Protein). To properly model promiscuous background, the control polypeptide must also be expressed to at least the same level as the bait protein, and should at least partially occupy the same intracellular locale. In cases where a protein of interest localizes to a specific subcellular location, it may also be useful to include an enzyme fusion that is specifically localized to this structure."
On top one should apply the general principles of good experimental design (replication, blocking and randomisation) as outlined for the AP-MS workflow.
Expected Outcome
"Proximity-dependent biotinylation approaches performed on a single bait protein can generate hundreds to thousands of putative partner identifications (with parameters such as the amount of material and the sensitivity of the mass spectrometer significantly influencing these numbers). Importantly, however, in most cases the majority of these proteins are not true proximity partners for the bait of interest. Many are instead: biotin-labeled in the absence of the recombinant enzyme (e.g. endogenously biotinylated proteins such as mitochondrial carboxylases); proteins that are promiscuously biotinylated with most baits (e.g. with BioID in HEK293 cells, filamin A belongs to this category), or; proteins that bind non-specifically to the affinity support (sepharose or other bead types)."
Proteome-scale proximity-dependent biotinylation examples
- Go, C. D. et al. A proximity-dependent biotinylation map of a human cell. Nature 595, 120–124 (2021). https://www.nature.com/articles/s41586-021-03592-2
Benchtop protocols
BioID
- Hesketh, G. G., Youn, J.-Y., Samavarchi-Tehrani, P., Raught, B. & Gingras, A.-C. Proteomics, Methods and Protocols. Methods Mol Biology 1550, 115–136 (2017) https://doi.org/10.1007/978-1-4939-6747-6_10
- describes the parallel implementation of both BioID and FLAG AP-MS allowing simultaneous exploration of both spatial and temporal aspects of protein interaction networks.
APEX2
- Hung, V., Udeshi, N., Lam, S. et al. Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat Protoc 11, 456–475 (2016). https://doi.org/10.1038/nprot.2016.018
- Benedict Tan, Suat Peng, Siti Maryam J.M. Yatim, Jayantha Gunaratne, Walter Hunziker, Alexander Ludwig, An Optimized Protocol for Proximity Biotinylation in Confluent Epithelial Cell Cultures Using the Peroxidase APEX2, STAR Protocols, Volume 1, Issue 2, 2020, 100074, ISSN 2666-1667, https://doi.org/10.1016/j.xpro.2020.100074