Results Realized: Better Understanding the Structure of CA125

Women’s Health Research at Yale’s 2023 Pilot Project led by Yale chemist and investigator Stacy Malaker, PhD, is now complete. "Developing Early Detection Diagnostic Tools in Ovarian Cancer" was made possible by the Wendy U. and Thomas C. Naratil Pioneer Award, an endowed fund of the Pilot Project Program. Created by Yale College Class of 1983 graduates, Wendy and Tom Naratil, the award provides up to $50,000 for research into women’s health each year.

Ovarian cancer is the nation’s deadliest gynecological cancer, often diagnosed late. Because symptoms are difficult to decipher from other common conditions such as bloating, pelvic pain, and urinary urgency, many cases are not caught until an advanced stage, when treatment options are few.

To save more lives, Malaker and her team pursued a new approach to understanding biomarkers—indicators of disease in the body—that can effectively detect ovarian cancer at an early stage, where survivability jumps to almost 95%.

"Not only do I have a deeply personal connection, I have always been passionate about women’s health and committed to researching the biochemistry of conditions and disorders that affect half of the nation’s population," says Malaker.

One human cell contains tens of billions of molecules: DNA, RNA, proteins, lipids, and carbohydrates, plus water and ions essential for chemical reactions and maintaining the cell’s environment. These molecules harmoniously work together to make cells, tissues, and ultimately entire organisms function.

CA125 is one single but very large and complex protein molecule. First identified in 1981, CA125 has been used as a biomarker for ovarian cancer for more than four decades. At the core of her Women’s Health Research at Yale Pilot Project, Malaker and the Malaker Lab, comprised of more than a dozen researchers, sought to examine CA125 in a very different way with the ambition of creating a more precise early diagnostic tool.

The CA125 protein sits on the surface of ovarian cancer cells, sheds, and circulates in the blood, becoming detectable through blood tests. However, CA125 levels naturally vary among women and are constantly changing, so a mere presence or certain CA125 value is not in and of itself a determination or a reliable indicator for early-stage ovarian cancer.

CA125 is 80% sugar by mass, making it a mucin, a very specialized and high-molecular-weight glycoprotein. The presence of sugar on CA125 is difficult to study given its dense structure and challenges with removal, paired with the fact that sugars invariably change in nearly every disease. Together, this causes a significant gap in foundational knowledge on that specific protein type.

Malaker is an expert in mass spectrometry—a powerful technique scientists use to measure and identify molecules—and one of a handful of chemists examining glycoproteins at this magnitude. The Malaker Lab isolates glycoproteins and leverages one of their three mass spectrometers to better understand what the proteins are made of, to their most basic forms: amino acid and monosaccharide units.

But before Malaker and team can understand what these glycoproteins look like at the molecular level, they needed to digest and isolate the desired components first. One of the biggest breakthroughs of this study was pioneering a new workflow to accomplish just that.

The team innovated GlycoFASP (Filter-Aided Sample Preparation)—a novel process to achieve enrichment through digestion utilizing filters. Here, enrichment means selectively isolating and concentrating certain molecules in order to better detect and analyze them. The protocol first depletes the CA125 sample of all contaminants, electrolytes, lipids, and protein fragments via centrifugation—high-speed spinning—using a filter. Next, enzymes called proteases are introduced to the refined CA125 sample and digest proteins. Finally, the sample flows through a filter, leaving behind highly pure glycopeptides, which are then ready for mass spectrometric analysis.

Incredibly, preparing the sample for analysis can be completed in one day, using less than one milligram of sample, in this instance blood serum. This paves the way for future ovarian cancer sample analysis. A paper detailing this groundbreaking workflow is currently under review for publication by a leading chemistry journal.

Now, with new knowledge about how to both effectively enrich specific molecules from complex samples and optimize a workflow to isolate measurable protein in CA125, the Malaker Lab has much more to discover. Malaker and her team are well on their way for additional learnings about the ways glycoproteins influence disease, establishing a diagnostic tool that can better predict ovarian cancer, and perhaps even future therapeutics, all building on the foundational findings from this Women’s Health Research at Yale Pilot Project. One of Malaker’s ultimate goals is to develop a blood test that can confidently be used by doctors to detect ovarian cancer in the earliest stage possible.

Also stemming from this Pilot Project were two additional journal articles published in Molecular and Cellular Proteomics (2025) and Analytical Chemistry (2025), as well as another under review. Malaker delivered more than a dozen presentations and seminars in the United States and around the world directly connected to this project as well.

Finally, because of the pilot data and new methods generated from this project, more than $2.25 million has been infused into the Malaker Lab to continue to examine and sequence glycoproteins.

“Not only are we studying cancer, but my lab is investigating Alzheimer’s disease, cardiovascular disease, pregnancy and fertility, and many more, at the most basic, molecular levels. I’m passionate about women’s health because it’s long been underfunded and there’s simply so much we still need to learn,” says Malaker. “I’m grateful to Women’s Health Research at Yale for investing in me and my lab to dedicate time, personnel, and resources to study CA125. As a result, we have much larger grants and tremendous momentum pushing discovery forward.”