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GMU Team Combines Protein Dyes and Mass Spec to Profile Protein Interaction Points

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NEW YORK (GenomeWeb) – Researchers at George Mason University have devised a mass spec-based method for identifying contact points involved in protein-protein binding.

Detailed this month in a paper published in Nature Communications, the technique uses dyes bound to the surface of interaction proteins to isolate their binding sites, which can then be analyzed using mass spectrometry.

Using the technique, the researchers identified contact points involved in the three-way interaction of the Interleukin-1β ligand, the receptor IL1RI, and the protein IL1RAcP. With this information, they then created peptide- and antibody-based inhibitors that targeted this interaction and abolished IL1β signaling.

The results open up the possibility that the technique could be useful in drug development research targeting such interactions, GMU researcher Alessandra Luchini, first author on the paper, told ProteoMonitor.

Such drugs, she said, "are very interesting to the pharmaceutical community, but [they] are very difficult to find using classical drug discovery [techniques]."

The technique relies on dyes that bind to protein surfaces with high affinity and low off-rates and block trypsin cleavage sites. Researchers apply complexes of interacting proteins with these dyes, which coat all portions of the proteins except those that are blocked because they are in contact with each other. They then denature the proteins and subject them to trypsin digestion. Because all portions of the proteins save for their interaction points have been coated in the dye, only those portions containing the interaction points are still accessible to trypsin, and so, only fragments from those regions of the protein will be generated in digestion.

These fragments can then be analyzed by mass spec to identify the portions of the target proteins involved in their interaction.

Of course, because native proteins are typically folded, there are potentially regions not involved in the interactions under investigation that will nonetheless be inaccessible to the dyes. To correct for this, the researchers also run simultaneous analyses of the proteins as they exist when not interacting with one another. By taking into account the peptides that are produced by digestion of these proteins, they can indentify which peptides are coming from the interaction sites, GMU researcher Lance Liotta, senior author on the paper, told ProteoMonitor.

As Liotta noted, there are several existing methods for identifying protein regions involved in protein-protein interactions, including cross-linking chemistry, which uses molecules to crosslink proteins at different points and mass spec to generate structural information based on the position of these linkages; and hydrogen/deuterium exchange, which involves labeling the amides in protein backbones with deuterium. Amides from the regions involved in binding will, in theory, be less accessible to labeling than those from other regions, and this can be detected using mass spec.

In the Nature Communications study, the researchers compared their technique to both crosslinking and HDX, finding, they wrote, that their dye-based method provided an overall higher number of positive hits and a higher percentage of true positives. The IL1β binding domain to which they developed peptide and antibody inhibitors was not identified by either crosslinking or HDX, they noted.

Liotta said the idea for the technique came from the researchers' contemplation of the fact that while dyes are commonly used in protein research for purposes like staining gels and immunohistochemistry, many protein-binding dyes haven't been explored for research purposes.

"We thought about all the dyes that are used to stain wool or other protein fabrics or dyes used in printing," he said. "There are a lot of dyes out there that have a very high affinity for proteins, but no one has been studying them as protein chemists, they've just been studying them as dyes for fabrics and other uses."

With this as their starting off point, the GMU team "set out to get our hands on all the kinds of dyes that we could find that were used in processes where the dye might bind to protein," Liotta said.

Ultimately, they identified a series of dyes that, when combined, allowed them to block all the trypsin sites on their target proteins. Typically, blocking all of the trypsin sites of a complex of interest requires a combination of between two and three dyes, Liotta said.

The GMU researchers have patented the dyes and the process and are currently looking for industrial partners both to commercialize the technique and to continue development of inhibitors they have developed using their findings, he said.

Another potentially interesting use for the dyes could be in middle-down proteomics research where they could be used to selectively block trypsin cleavage sites to allow for the creation of larger peptides for mass spec analysis, Liotta said.

By using peptides larger than those in traditional bottom-up proteomics but smaller than intact proteins, middle-down proteomics hopes to simplify and improve analysis of features like PTMs and enable more complete sequence coverage.

As Albert Heck, chair of the Biomolecular Mass Spectrometry and Proteomics group at Utrecht University, told ProteoMonitor in a recent interview on the subject, "If you can cut a protein in five pieces, then you only have to analyze these five stretches and then you have 100 percent sequence coverage. So you don't have to be Einstein to think that that is a better approach than cutting it into 50 pieces."

The most significant difficulty facing middle-down researchers at the moment is how to chop proteins into this desired size. Currently, there is no protease ideal for such work, and while researchers are exploring various chemical methods, these are often complicated by side chain reactions and oxidations.

Liotta suggested that the various dyes used for his and his colleagues' protein-interaction work could also prove useful as reagents for middle-down proteomics.

"You could characterize the types of trypsin consensus sequences that [a given dye] likes to stick to, so it would be feasible to use one dye to knock out, say, a third of your trypsin sites," he said.

In an email to ProteoMonitor, Yury Tsybin, a professor at Ecole Polytechnique Fédérale de Lausanne and a leading middle-down researcher, said that the dye-based approach was potentially interesting. However, he noted, it is unclear whether or not it will allow for reproducible digestion or whether the dye itself might interfere with mass spec analysis.

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