Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches

Payman Samavarchi-Tehrani,Anne-Claude Gingras,Reuben Samson

doi:10.1074/mcp.r120.001941

Abstract

The study of protein subcellular distribution, their assembly into complexes and the set of proteins with which they interact with is essential to our understanding of fundamental biological processes. Complementary to traditional assays, proximity-dependent biotinylation (PDB) approaches coupled with mass spectrometry (such as BioID or APEX) have emerged as powerful techniques to study proximal protein interactions and the subcellular proteome in the context of living cells and organisms. Since their introduction in 2012, PDB approaches have been used in an increasing number of studies and the enzymes themselves have been subjected to intensive optimization. How these enzymes have been optimized and considerations for their use in proteomics experiments are important questions. Here, we review the structural diversity and mechanisms of the two main classes of PDB enzymes: the biotin protein ligases (BioID) and the peroxidases (APEX). We describe the engineering of these enzymes for PDB and review emerging applications, including the development of PDB for coincidence detection (split-PDB). Lastly, we briefly review enzyme selection and experimental design guidelines and reflect on the labeling chemistries and their implication for data interpretation.

Highlights

The study of protein subcellular distribution, their assembly into complexes and the set of proteins with which they interact with is essential to our understanding of fundamental biological processes
Complementary to traditional assays, proximity-dependent biotinylation (PDB) approaches coupled with mass spectrometry have emerged as powerful techniques to study proximal protein interactions and the subcellular proteome in the context of living cells and organisms
The full applicability of these approaches is not yet clear: for antibody-based approaches, issues of antibody cross-reactivity will need to be addressed whereas the ivBioID technique is likely to be more useful for proteins that remain tightly anchored to a structure following permeabilization

Summary

In Brief

Proximity-dependent biotinylation approaches such as BioID and APEX overcome classical limitations of biochemical purification and have gained widespread use in recent years for revealing cellular neighborhoods. Complementary to traditional assays, proximity-dependent biotinylation (PDB) approaches coupled with mass spectrometry (such as BioID or APEX) have emerged as powerful techniques to study proximal protein interactions and the subcellular proteome in the context of living cells and organisms Since their introduction in 2012, PDB approaches have been used in an increasing number of studies and the enzymes themselves have been subjected to intensive optimization. In the past 8 years, alternative approaches have been introduced that instead bypass the requirement to maintain protein-protein interactions or organellar integrity during sample purification These are referred to as proximity-dependent biotinylation (PDB) approaches and consist of directing an enzyme capable of catalyzing covalent transfer of biotin (or other derivatives) to endogenous proteins that are located within a certain distance of the enzyme.

Proximity Dependent Biotinylation Enzymes for Proteomics

OVERVIEW OF PDB ENZYMES

IMPROVEMENT OF PDB ENZYME CHARACTERISTICS

DEVELOPMENT OF NEW PDB MODALITIES

CONSIDERATIONS FOR EXPERIMENTAL DESIGN

PROXIMAL LABELING CHEMISTRY AND LABELING PROPENSITY

Findings

CONCLUSION