Discovering cellular protein‐protein interactions: Technological strategies and opportunities

Abstract

The analysis of protein interaction networks is one of the key challenges in the study of biology. It connects genotypes to phenotypes, and disruption often leads to diseases. Hence, many technologies have been developed to study protein-protein interactions (PPIs) in a cellular context. The expansion of the PPI technology toolbox however complicates the selection of optimal approaches for diverse biological questions. This review gives an overview of the binary and co-complex technologies, with the former evaluating the interaction of two co-expressed genetically tagged proteins, and the latter only needing the expression of a single tagged protein or no tagged proteins at all. Mass spectrometry is crucial for some binary and all co-complex technologies. After the detailed description of the different technologies, the review compares their unique specifications, advantages, disadvantages, and applicability, while highlighting opportunities for further advancements.

Emerging roles of allosteric modulators in the regulation of protein‐protein interactions (PPIs): A new paradigm for PPI drug discovery

“…Extensive efforts have been invested in the development of experimental methods for the detection of PPIs, [56][57][58][59][60][61] whose theoretical basis can be mainly categorized into genetics, biophysics, and proteomics. One of the classic examples of detection methods utilizing genetic techniques is the yeast two-hybrid system.…”

“…68 Mass spectrometry (MS)-based proteomics has also been used to explore PPIs. 57,61,[69][70][71] In this process, a specific tag such as FLAG-tag or HA-tag is first attached to the protein of interest, which is referred to as the "bait". Using an anti-tag system on an immobilized solid support, the tagged protein and its interaction partners can be purified.…”

“…There are various optimized versions of MS-based methods, such as tandem affinity purification MS, affinity enrichment MS, and immunopurification MS, which supply comprehensive insights into the dynamic PPI networks. 61,69,71,74,75 As a powerful tool for PPI detection, especially at the level of protein complexes, MS has been widely applied for research regarding the interactome and proteome. [76][77][78][79] Despite these successes, there remain limitations for MS-based approaches, such as the relatively large requirement for sample input, and further miniaturizations and optimizations are still underway.…”

“…Additionally, there are various optimized versions of BRET, such as BRET n (NanoLuc luciferase‐based bioluminescence resonance energy transfer), which utilizes the highly sensitive NanoLuc luciferase as the donor and enables improved sensitivity and efficiency in PPI detection . Mass spectrometry (MS)‐based proteomics has also been used to explore PPIs . In this process, a specific tag such as FLAG‐tag or HA‐tag is first attached to the protein of interest, which is referred to as the “bait”.…”

“…MS‐based PPI detection exhibits high specificity and an ability to identify spatially and temporally dynamic PPI complex systems. There are various optimized versions of MS‐based methods, such as tandem affinity purification MS, affinity enrichment MS, and immunopurification MS, which supply comprehensive insights into the dynamic PPI networks . As a powerful tool for PPI detection, especially at the level of protein complexes, MS has been widely applied for research regarding the interactome and proteome .…”