New method allows researchers to detect proteins at close range in single cells

Currently, most methods for identifying proteins inside a cell are based on a rough census; Scientists often grind up a large batch of cells before characterizing their genetic material. But just as a population of 100 single individuals differs in many ways from a population of 20 households of five, such a description fails to gather information about how proteins interact and aggregate into functional groups.

Now, researchers at the University of Chicago’s Pritzker School of Molecular Engineering (PME) have developed an approach that allows them to more easily examine whether proteins are close together within a cell. The technology, which can be performed simultaneously with more routine gene sequencing, is described in the journal. Nature Methods.

“This is a modern, high-throughput way of looking at protein functions inside individual cells,” said Savaş Tay, professor of molecular engineering and senior author of the new study. “I think this method will be an important resource for the molecular biology community.”

Function oriented

In recent years, with the advent of rapid and inexpensive genetic sequencing technologies, scientists have turned to single-cell mRNA sequencing to obtain snapshots of the cell’s internal states. By sequencing all of a cell’s mRNA molecules that encode proteins, they can get an idea of ​​what proteins a cell might be actively using. But such a surrogate for protein abundance does not tell the whole story.

“Just knowing the abundance of certain proteins doesn’t always tell you about how a cell works,” Tay said. “Often, whether proteins are functionally active is not just about whether they are present but whether they form complexes.”

Tay wanted to capture whether the proteins were physically close together, and he wanted to do this in a fast, high-throughput way, not by relying on microscopy to visualize their positions or by isolating a few proteins at a time to examine them more closely.

He and his colleagues developed molecular probes with outwardly extending DNA tags that bind to proteins of interest. If two proteins are physically close to each other, these DNA probes stick together like Velcro. In this way, researchers can set up experiments that simultaneously investigate hundreds of proteins. They then use routine sequencing to read back the DNA probes that are linked together and determine which proteins are paired.

“In the vast space inside a cell, it’s very unlikely that two probes will find each other unless they are attached to nearby proteins,” Tay said. “When the probes attach, this tells us that these proteins are very close together.”

Because the approach, called prox-seq, uses standard sequencing, researchers can analyze a cell’s mRNA simultaneously with related proteins, helping answer questions about the correlation between mRNA and protein abundance and function.

proof of concept studies

To demonstrate the benefit of Prox-seq, Tay’s team first tested it on two subsets of human immune cells, B cells and T cells. The technique they found was only able to differentiate cells based on what protein-protein interactions are present in each cell type. It can also identify previously unknown subsets of cells based on slight differences in how groups of proteins are organized within cells. Using human-derived peripheral blood mononuclear cells, they simultaneously measured 38 individual proteins, 741 protein complexes, and thousands of mRNAs in each cell, and discovered a new protein complex that identified naive T cells.

Next, the group used Prox-seq to examine how proteins change regulation in another type of immune cell, macrophages, when cells are activated in response to a pathogen. Once again, they identified both known interacting protein pairs and novel protein groups that could be used to determine not only whether a macrophage has been activated but also what type of pathogen it has been exposed to.

“This showed that our method not only validates protein-protein complexes that we already know, but can also find new interactions between proteins,” Tay said.

So far, the researchers have only tested the method on proteins found on the surface of cells; They are now working to expand it to more protein types, while developing ways to decipher exactly where a cell’s proteins interact.