Current Approaches Toward Quantitative Mapping of the Interactome

Abstract

Protein-protein interactions (PPIs) play a key role in many, if not all, cellular processes. Disease is often caused by perturbation of PPIs, as recently indicated by studies of missense mutations. To understand the associations of proteins and to unravel the global picture of PPIs in the cell, different experimental detection techniques for PPIs have been established. Genetic and biochemical methods such as the yeast two-hybrid system or affinity purification-based approaches are well suited to high-throughput, proteome-wide screening and are mainly used to obtain qualitative results. However, they have been criticized for not reflecting the cellular situation or the dynamic nature of PPIs. In this review, we provide an overview of various genetic methods that go beyond qualitative detection and allow quantitative measuring of PPIs in mammalian cells, such as dual luminescence-based co-immunoprecipitation, Förster resonance energy transfer or luminescence-based mammalian interactome mapping with bait control. We discuss the strengths and weaknesses of different techniques and their potential applications in biomedical research.

Keywords: BRET; BiFC; DULIP; FCCS; FRET; Interactome Mapping; LUMIER; PLA; PPI analysis; Quantification of protein-protein interactions.

FIGURE 1

Overview of genetic protein–protein interaction (PPI) methods. (A) In Fluorescence cross-correlation spectroscopy (FCCS) measurements, co-migration of two fluorescently labeled molecules through a focal volume is quantified. (B) bimolecular fluorescence complementation (BiFC) utilizes two non-fluorescent fragments of EGFP or a variant. Upon interaction of the two labeled proteins, the fragments can reassociate, resulting in fluorescence. (C) The principle of bimolecular luminescence complementation (BiLC) is similar to BiFC but is based on two fragments of a luciferase. In contrast to BiFC, the reassociation is reversible. (D) Close proximity of two DNA oligomer-labeled antibodies allows circularization of two additional oligomers after hybridization. The product is amplified in a rolling circle reaction and subsequently detected with fluorescently labeled probes. (E) During Förster resonance energy transfer (FRET), energy is transferred non-radiatively from an excited donor molecule to an acceptor molecule. In case the acceptor is also a fluorophore, the transmitted energy is emitted at a longer wavelength (the so called sensitized emission). (F) bioluminescence resonance energy transfer (BRET) is similar to FRET with the difference that a luciferase serves as a donor molecule. (G) In dual luminescence-based co-immunoprecipitation (DULIP) assays, two proteins of interest are fused to firefly or Renilla luciferase, respectively. An additional PA-tag allows precipitation of the bait protein from the lysate. If an interaction occurs, co-precipitation of the prey protein is indicated by luminescence arising from the firefly luciferase.