Protein-Protein Interaction Networks Service

What Is Protein-Protein Interactions In Proteomics?

Enormous molecular processes are regulated via a large number of protein components organized by Protein-Protein Interactions, which refer to intentional physical contacts established between two or more proteins and resulted in specific biochemical events. Such interactions undertake at the core of the entire interactomics system of the living cells, unsurprisingly, specific PPIs are identified with a correlation of multiple diseases.

Protein-protein interactions form the basis of life phenomena, and studying these interactions can elucidate the mechanisms behind biological responses and reveal the essence of life phenomena. While some proteins can exert their functions as monomers, the majority of proteins function through protein-protein interactions or by forming protein complexes. Therefore, investigating protein interactions has become an essential aspect of studying protein function and mechanisms of action.

If we want to study whether one protein interacts with another protein or with several other proteins, the first step is to identify the target protein and then identify other associated proteins. This process ultimately enables us to achieve our experimental objectives.

Protein Protein Interaction Detection Technique

In Vivo Detection Method:

Co-IP

Co-IP combined with Mass Spectrometry (MS), often referred to as Co-IP-MS or IP-MS (Co-Immunoprecipitation Mass Spectrum, Immunoprecipitation Mass Spectrum), is an important tool for discovering and studying protein-protein interactions. Co-IP-MS or IP-MS is a classical method based on the specific interaction between antibodies and antigens, used to investigate protein-protein interactions. It is an effective approach for determining the physiological interactions between two proteins within intact cells.

When cells are lysed under non-denaturing conditions, many protein-protein interactions present inside the cells are preserved. By using specific antibodies or other affinity reagents to bind to the target protein, Co-IP extracts the target protein along with its interacting proteins from complex samples. Subsequently, mass spectrometry is employed to identify the interacting partners of the target protein. SILAC technology combined with Co-IP-MS technology can be used for high-throughput quantitative analysis of protein interactions networks in specific cases. (For a detailed introduction to the Co-Immunoprecipitation experiment, please refer to the Co-IP experimental protocol.)

FRET

Fluorescence Resonance Energy Transfer (FRET) is an effective method used for qualitative and quantitative detection of interactions between biomacromolecules. Depending on the configuration of the fluorescence microscope, FRET can be applied at various levels, such as in solution, cell suspensions, multicellular systems, single cells, cell membranes, and cellular organelles, to study interactions, distances, and kinetic properties between biomacromolecules.

In the field of life sciences, FRET technology is a powerful tool for detecting nanoscale distances and nanoscale distance changes between biomacromolecules in living organisms. It can be used to determine whether two protein molecules directly interact within a single cell. As mentioned earlier, FRET occurs when the emission spectrum of the donor fluorophore overlaps with the absorption spectrum of the acceptor fluorophore, and the distance between the two probes is within the range of 10 nm. In biological systems, if the distance between two protein molecules is within 10 nm, it is generally considered that these two protein molecules have a direct interaction.

TAP- Mass Spectrometry

Regardless of which tag is used as the bait protein to pull down the target protein, it can only serve as a crude enrichment step. When the relationship between the proteins is known or predicted before the experiment, Western Blotting can be used for validation. However, when discovering a new, unknown interaction, a high-throughput, high-resolution technique is required, and this is where mass spectrometry comes into play.

The steps of affinity purification are similar to Co-IP, but single-tag purification efficiency is low, and there may be a higher amount of residual non-specific proteins. Therefore, TAP has been developed, which utilizes dual-tag purification. TAP can use two different tags in combination, but it is essential to note that no tag combination is universally ideal. The choice of tags mainly depends on their characteristics, the properties of the target protein, such as stability and solubility, and the fusion compatibility between the tag and the target protein. The protein complex obtained from affinity purification is then subjected to mass spectrometry technology for identification and analysis.

BioID-MS

Proximity labeling through the use of modifying enzymes is a novel PPI screening method that enables the detection of transient and weak interactions. By fusing the modifying enzyme with the protein of interest, it can label nearby and potential interacting proteins. This type of technique includes BioID, a selective proximity labeling method for the proteome. Compared to traditional protein interaction discovery methods, BioID technology has significant advantages and is suitable for identifying transient, weak interactions, insoluble cellular structures, and dynamic processes within organisms.

TurboID

Compared to BioID, TurboID has higher catalytic efficiency and lower temperature requirements. TurboID can biotinylate neighboring proteins within a range of approximately 10nm in live cells. Finally, the biotinylated proteins are enriched and purified using streptavidin-coupled magnetic beads, followed by mass spectrometry analysis to identify the neighboring proteins of the target protein in the physical space.

In Vitro Detection Method

Glutathione S-Transferase (GST) Pull-down

GST pull-down is a technique similar to Co-IP, but with a difference that GST pull-down is used for studying protein interactions in vitro, while Co-IP is more commonly used for intracellular interactions. In this method, a fusion protein containing a GST-Tag is used to pull down interacting proteins. The fusion protein is then immobilized and precipitated using glutathione resin beads that can specifically bind to the GST tag. Subsequently, SDS-PAGE is used to separate the protein complexes. Similarly, other tags such as Strep, FLAG, MYC, etc., can also be added to the proteins. However, this method cannot reveal the natural interactions that occur between proteins. (Detailed GST pull-down technology introduction)

Far-Western Blotting

Far-Western blotting is a method derived from Western blotting, specifically designed for studying protein-protein interactions. This technique utilizes labeled or antibody-detected "bait" proteins to detect "prey" target proteins on a transfer membrane. If the bait protein interacts with the target protein, specific bands can be detected using the specificity of the bait protein. Far-Western blotting has been employed to study interactions between proteins involved in DNA replication and repair, and it is considered a rapid, efficient, and reproducible method for investigating protein-protein interactions.

This method allows for the detection of direct interactions between two proteins. If the interaction is indirect, further testing can be conducted using candidate proteins. Due to its high sensitivity, Far-Western blotting has found wide applications in various research fields. (For a detailed introduction to Far-Western blotting technique, please refer to the Far-Western blot experimental protocol.)

DARTS

DARTS (Drug Affinity Responsive Target Stability) is a new technique developed based on the reduced sensitivity of target proteins to protease degradation caused by the binding of small molecule drugs to their target proteins. This technique can be used for drug screening and target identification without chemical modification of the compound, as well as not dependent on drug activity.

SPR

Surface plasmon resonance, or SPR, has been used to characterize and measure interactions between biomolecules since it was first applied to the study of processes on metal surfaces and gas sensing in the early 1980s. The number of publications employing SPR biosensors for medical diagnostics, environmental monitoring, food safety, and security-related analyses has grown, as has the development of SPR sensors for detecting chemical and biological species.

Surface plasmon resonance principle

This imaging technology provides spatial resolution for protein interactions, allowing detailed studies of proteins using high-throughput microarray techniques. It enables multiplex detection of different proteins without compromising sensitive regions. Fluorescence detection is a widely used imaging technique that allows visualization of proteins labeled with fluorescent dyes. However, label-free techniques are also essential as they eliminate potential impacts of labeling on protein function.

It is crucial to note that unlike label-based methods, label-free techniques directly measure the intrinsic physical properties of proteins, providing additional information such as the mass and size of each protein. Surface Plasmon Resonance Imaging (SPRi) is a label-free in situ detection and molecular binding study technique.

Protein Microarrays

Protein microarrays are chips or slides where various proteins are systematically immobilized on a solid support surface. Samples containing labeled or fluorescently tagged proteins are hybridized with the probe proteins on the microarray. Unbound proteins are washed away, and the fluorescence intensities at each spot on the microarray are measured using a fluorescence scanner or confocal laser scanning technology. By analyzing the fluorescence intensities, protein-protein interactions can be studied, ultimately leading to the determination of protein functions. This method is rapid, simple, and high-throughput, allowing the detection of low abundance and low molecular weight proteins that are often challenging to identify. However, the specificity of binding to the target protein needs improvement.

Phage Display

Phage display technology is a laboratory technique used to study protein-protein, protein-peptide, and protein-DNA interactions. It involves using bacteriophages (viruses that infect bacteria) to link proteins to their genetic information. In this technique, the gene encoding the target protein is inserted into the bacteriophage coat protein gene, allowing the bacteriophage to display the protein on its exterior while containing the gene for the protein inside, thus establishing a link between genotype and phenotype. Other proteins, peptides, or DNA sequences can then be screened against these displayed bacteriophages to detect interactions with the displayed protein. Through this process, large protein libraries can be screened and amplified in a process similar to natural selection, known as in vitro selection.