Today, most methods to determine the proteins inside a cell rely on a crude census—scientists usually grind a large group of cells up before characterizing their genetic material. But just as a population of 100 single people differs in many ways from a population of 20 five-person households, this kind of description fails to capture information about how proteins are interacting and clumping together into functional groups.
Now, researchers at the University of Chicago’s Pritzker School of Molecular Engineering (PME) have developed an approach that lets them more easily study whether proteins are located in close proximity to each other inside a cell. The technology, which can be carried out at the same time as more routine gene sequencing, is described in the journal Nature Methods.
“This is a streamlined and high-throughput way to look into protein functions inside individual cells,” said Savas Tay, professor of molecular engineering and senior author of the new work. “I think this method is going to be a major resource for the molecular biology community.”
Focused on function
In recent years, with the advent of fast and cheap genetic sequencing technologies, scientists have turned to single-cell mRNA sequencing to get snapshots of cell’s internal states. By sequencing all of a cell’s mRNA molecules—which encode proteins—they can get an idea of what proteins a cell might be actively using. But this kind of proxy for protein abundance doesn’t convey the full story.
“Just knowing the abundance of certain proteins doesn’t always give you information about how a cell is functioning,” said Tay. “Often, whether or not proteins are functionally active has to do with not just whether they are present but whether they are forming complexes.”
Tay wanted to capture whether proteins were physically near each other—and do so in a fast, high-throughput way, not by relying on microscopy to visualize their locations or isolating a few proteins at a time for closer inspection.
He and his colleagues developed molecular probes that attach to proteins of interest and have DNA tags extending outward. If two proteins are physically close to each other, those DNA probes stick together like Velcro. The researchers can set up experiments that simultaneously probe hundreds of proteins in this way. Then, they use routine sequencing methods to read back any DNA probes that bound to each other and determine which proteins were paired up.