New biosensor technology maps enzyme mystery inside cells

Cornell researchers have developed a powerful new biosensor that reveals, in unprecedented detail, how and where kinases - enzymes that control nearly all cellular processes - turn on and off inside living cells.

The advance provides scientists with a new way to study the molecular switches that regulate cellular processes, including cell growth and DNA repair, as well as cellular responses to chemotherapy drugs and pathological conditions such as cancer.

Cells rely on kinases to control processes from cellular metabolism and growth to stress responses. Unraveling how the more than 500 kinases in human cells all work together is one of biology's biggest puzzles. Until now, researchers lacked robust tools to see exactly where and how these enzymes act inside cells. Understanding those precise signaling patterns is key to learning how cells respond to drugs - and to designing more effective therapies.

The new technique, called ProKAS (Proteomic Kinase Activity Sensors), is described in a studypublished Nov. 13 in Nature Communications by the lab of Marcus Smolka, professor of molecular biology and genetics in the College of Agriculture and Life Sciences and at the Weill Institute for Cell and Molecular Biology.

"Given the challenges with current technologies, it was important that we completely reimagined the way we can read kinase activity and provide spatial resolution," said Smolka, associate vice provost in Cornell Research and Innovation. "In this case, we use mass spectrometry to read the activity, and this is a different approach to tracking kinase action in cells compared to microscopy-based techniques currently used."

ProKAS works by using chains of amino acids, known as peptides, engineered to imitate the natural proteins kinases act on. Each peptide carries a unique amino acid "barcode" that marks its location within the cell. When a kinase acts on the peptide, mass spectrometry detects both the action and its corresponding barcode, revealing the kinase's activity, location and timing. This allows scientists to monitor many kinases at once, across multiple regions of a cell, with high precision and speed, creating a spatial map of enzyme activity.

For this study, Smolka's team used the barcoded peptides to monitor kinase activity during cells' response to a range of anti-cancer drugs that induce DNA damage.

"One of the key innovations here is the use of barcodes, which have been used in genomics to study DNA, but here we're applying them to proteins here for the first time," Smolka said. "The approach lets us follow multiple kinases at once and see exactly when and where they act inside cells, giving a level of detail in kinase signaling that hasn't been possible before."

Using ProKAS, the researchers were able to track the action of kinases that respond to DNA damage, to see exactly where and when they became active inside cells, including in specific parts of the nucleus. They observed how key DNA damage response kinases, such as ATR, ATM, and CHK1, reacted over time, revealing differences in activity across regions that could not be measured before. The system also handled many samples quickly, showing that it could be scaled up for larger studies.

Smolka said the team can already analyze 36 samples in a single 30-minute mass spectrometry run.

"We're already scaling up," said Will Comstock, Ph.D. '25, the first author on the paper and a former researcher in Smolka's lab. "We're going up to hundreds of samples, and the idea in the future is to be able to analyze several hundreds or even thousands."

ProKAS's design also makes it adaptable for studying other human kinases. In the future, the technology could help scientists explore poorly studied kinases and help pharmaceutical researchers identify new drugs that affect kinase activity in disease processes, Smolka said.

"For pharmaceutical companies that want to understand the impact of trial drugs, this would be a way to do that in a high-throughput fashion," Smolka said. "Researchers can rapidly screen and determine mechanisms of drug action. I think that has a huge value."

Looking ahead, the team plans to integrate ProKAS with computational design tools, expanded peptide libraries and other approaches to deepen understanding of how kinases shape cell behavior.

In addition to Comstock, co-authors are Deanna V. Maybee, Ph.D. '23, Yiseo Rho '26, doctoral student Mateusz Wagner and postdoctoral researcher Yingzheng Wang, all in the Smolka Lab; and research associate Marcos V.A.S. Navarro. The study was funded by the National Institutes of Health and the National Science Foundation.

Stephen D'Angelo is the communications manager for biological systems at Cornell Research and Innovation.