NEW YORK (GenomeWeb) – Proteomics firm Tymora Analytical Operations has developed a workflow for isolating extracellular vesicles (EVs) and analyzing their protein contents via mass spectrometry.
Described in a study published last month in the Journal of Proteome Research, the approach uses functionalized magnetic beads to capture EVs circulating in patient samples like urine or plasma and is particularly suited to the analysis of EV protein content, said Anton Iliuk, Tymora's president and chief technology officer and an author on the paper.
The West Lafayette, Indiana-based company is currently using the method to develop a urine-based test that measures EV-bound phosphoproteins to detect recurrence in patients treated for bladder cancer. It is also investigating EV phosphoproteomic signatures in breast cancer patients with the goal of using those signatures to assess the effectiveness of different targeted therapies.
Shed by cells into various bodily fluids, EVs are thought to reflect the molecular make-up of their cells of origin. This has made them an area of growing interest in liquid biopsy research, the idea being that it might be easier to measure proteins or nucleic acids in exosomes derived from, for instance, cancer cells, than to detect cancer-linked nucleic acids or proteins circulating freely in patient blood or urine.
A number of researchers and companies have identified EV-bound biomarkers linked to various diseases, with firms like Exosome DX (acquired this summer by Bio-Techne) developing tests to help clinicians diagnose prostate cancer and guide therapy in lung cancer.
However, much of the work looking at exosomes and other EVs has focused on nucleic acid markers, Iliuk noted, and those efforts that have looked at proteins have typically used antibody-based approaches that are not well-suited to broad, unbiased discovery work.
One challenge facing EV-based proteomics has been the lack of an EV enrichment method well-suited to proteomic analysis, Iliuk said. Differential centrifugation has been commonly used for isolating EVs from bodily fluids, but as he and his co-authors noted, it is a time-consuming and cumbersome process that is poorly suited to clinical work given its low reproducibility.
Researchers have since developed a variety of other approaches for isolating EVs but, the authors noted, they still have fairly low yields (in the 5 to 25 percent range) and, importantly for proteomic work, often isolate high-abundant circulating proteins along with the target EVs, contaminating the sample so far as protein analysis is concerned.
While such contaminants are not necessarily an issue for nucleic acid analyses, "for us contaminating proteins are indeed a big problem, because they will potentially suppress any signal from real, important targets we are looking for," Iliuk said.
Tymora's assay, which the company has named EVTRAP (Extracellular Vesicles Total Recovery And Purification) uses magnetic beads functionalized with hydrophobic and aromatic lipophilic groups that will bind to the lipid membranes encapsulating EVs while not capturing the high-abundance circulating proteins that have previously confounded proteomic analyses of EVs, Iliuk said.
In the JPR study, the researchers combined the EVTRAP approach with PolyMAC-based phosphopeptide enrichment to a 10-ml urine sample, identifying roughly 2,000 phosphopeptides from more than 860 proteins. Using ultracentrifugation coupled to the same LC-MS workflow, they identified 165 phosphopeptides from 104 proteins. Previous studies, the authors noted, had identified as few as 14 exosome phosphoproteins from as much as 400 ml of urine.
Looking at the non-modified proteome, the researchers were able to consistently detect more than 16,000 peptides from roughly 200 proteins from 200 µL of urine, compared to around 7,200 peptides from 1,100 proteins using ultracentrifugation.
Iliuk said Tymora has also demonstrated the EVTRAP approach in plasma, where it also "performs very well," despite plasma's considerably greater complexity. He and his colleagues are currently writing a manuscript detailing this work, he said.
The company is also working to develop a version of the assay compatible with a 96-well format, which could allow it to be easily automated, Iliuk said, noting that this would give it the high-throughput and reproducibility needed for clinical validation and, ultimately, actual clinical assays.
The company is collaborating with researchers at the Indiana University School of Medicine analyzing urine samples from patients with high-grade and low-grade bladder cancer to see if they can identify EV-bound proteins or phosphoproteins that could be used for monitoring bladder cancer patients for recurrence after treatment.
Such an assay could be useful given bladder cancer's high rate of recurrence, which is typically tested for using cystoscopy. Analysis of EV-bound proteins in urine could allow for an easier, non-invasive approach to patient monitoring, Iliuk said.
The company and its collaborators are also looking into whether EV-bound proteins in plasma could help monitor patient response to cancer immunotherapies as well as targeted treatments in breast cancer.
In general, the approach could prove useful for applications where clinicians would like to sample patients more frequently than they are able to currently or where testing requires invasive procedures like biopsies, Iliuk said. This is, of course, the rationale for liquid biopsies more broadly, but he said the company believes analysis of EVs could prove more effective than analytes like ctDNA or circulating proteins.
In particular, EVs provide researchers the opportunity to analyze phosphoproteins without having to obtain a tissue sample, something that Iliuk said he believes could boost the role of phosphoproteomics in clinical applications. Freely circulating phosphoproteins have been difficult to measure in patient biofluids due to the complexity of these samples and the presence of phosphatases that can alter their phosphorylation status.
Iliuk said he expected the company would partner with outside firms or out-license any clinical assays it develops using the technology, with the bladder cancer assay being its lead project currently. Tymora is also offering the EVTRAP-mass spec workflow as a service to companies and researchers looking to do EV biomarker discovery and validation work.