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Purdue Team IDs Breast Cancer-Linked Phosphoproteins in Extracellular Vesicles

NEW YORK (GenomeWeb) – A team led by Purdue University researchers has identified a set of candidate breast cancer biomarkers by analyzing the phosphoprotein content of circulating extracellular vesicles (EVs).

In a study published this week in the Proceedings of the National Academy of Sciences, the scientists identified 144 phosphoproteins in plasma EVs that were more highly phosphorylated in breast cancer patients compared to healthy controls.

In a targeted analysis measuring phosphorylation levels of four of these 144 proteins, they found in three of the four — RALGAPA2, PKG1, and TJP2 — significant differences between levels in breast cancer patients and controls. In the case of the fourth protein, NXF1, they observed a difference between the two patient populations, but this difference was notably smaller than what they observed in the initial discovery experiment and, ultimately, "statistically inconclusive," they wrote.

The effort fits into a broader trend within biomarker research toward exploring EVs like microvesicles and exosomes as sources of markers. One of the challenges of discovering and developing circulating biomarkers is the complexity of plasma, which makes reproducibly detecting disease-linked markers, which are often low abundance, a difficult analytical problem.

EVs represent a promising way forward in that they may contain proteins and other molecules useful in molecular tests, but in an environment far less complex than plasma. The PNAS researchers also noted that in the case of phosphoproteomics markers, EVs protect modified proteins from "proteases and other enzymes," which "make them highly stable in a biofluid for extended periods of time."

The Purdue team isolated exosomes and microvesicles from patient plasma samples (30 breast cancer patients and 6 healthy controls) using centrifugation, following this with mass spec analysis of their phosphoproteomic content using a Thermo Fisher Scientific Orbitrap Velos. This analysis identified 9,643 phosphopeptides from 2,413 proteins, making it the "largest group of EV phosphoproteins [identified] to date," the authors wrote.

From this set, they identified 144 proteins whose phosphorylation levels differed significantly between cases and controls, and, using parallel-reaction monitoring mass spec analysis on a Thermo Fisher Q Exactive HF, they confirmed these observed differences in the aforementioned three proteins.

The study also yielded data suggesting that protein phosphorylation could be more informative regarding breast cancer status than protein expression alone.

"Quantitative analyses of EV proteomes revealed strikingly similar expression of most proteins in healthy individuals and patients with cancer," the researchers noted. "In comparison, there are a larger number of phosphorylation sites with significant changes in patient samples."

As one of the most common cellular signaling processes, protein phosphorylation has been a large focus of cancer biomarker research, and kinases, the proteins responsible for protein phosphorylation, are a significant and growing area in drug development.

As the authors wrote, much additional work is needed to validate the markers identified in the study, including evaluation in larger patient cohorts including subjects with various breast cancer subtypes. It will also be necessary to improve the reproducibility of their EV isolation techniques, they noted.

That said, the study suggests that EVs could prove a source of phosphoproteomics biomarkers and that it is possible to isolate and measure thousands of phosphoproteins from these structures.