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New Glycoprotein Array May Enable Improved Cancer Biomarker Detection


NEW YORK (GenomeWeb) – Researchers from Purdue University have developed a new reverse phase protein array (RPPA) platformfor the capture and analysis of glycoproteins.

Described in a paper published last week in the Journal of the American Chemical Society, the platform, which they named  polymer-based reverse phase glycoprotein array (polyGPA), allows users to detect and quantify glycoproteins with improved sensitivity and specificity compared to existing methods, Andy Tao, professor of biochemistry at Purdue and senior author of the publication, told GenomeWeb.

Tao plans to commercialize the technology through a biotech firm, Tymora Analytical Operations, that he and his colleagues launched in 2010. To date, the company's work has focused primarily on phosphoproteome analysis.

Glycoproteins are key molecules in biomarker research, comprising more than half of current US Food and Drug Administration-approved protein cancer markers and around 80 percent of all FDA-approved protein markers currently in clinical use.

Nonetheless, glycoproteome research has struggled due to the great complexity of protein glycosylation patterns and a lack of good tools for such analyses. For instance, as Tao and his co-authors observed, "glycosylation-specific antibodies are virtually nonexistent." Additionally, they noted, "the high complexity of thousands of proteins in an unenriched mixture makes it extremely challenging to detect specific glycoproteins."

Mass spectrometry has proven a popular method of glycoproteome analysis. However, due to the enrichment and sample prep steps commonly used for such analyses, relatively large samples are often required, and this can limit the method's applicability for studying glycoproteins in cases where sample size is limited, as in certain tumor biopsies, for instance.

To tackle these challenges, Tao and his colleagues combined RPPA technology with arrays functionalized for glycoprotein capture, allowing them to look specifically at the glycosylated subset of the proteome while using conventional antibodies.

RPPA uses cell lysates spotted in array format that can then be probed with antibodies to multiple proteins of interest. The technique has been used extensively for phosphoproteome analysis, employing antibodies specific to phosphorylated forms of proteins of interest.

By functionalizing their RPPAs to enrich for glycoproteins, the Purdue researchers eliminated the need for glycoprotein-specific antibodies. Because their array captures only glycoproteins, they can probe their samples with antibodies to the generic form of the proteins, knowing that whatever proteins they detected and quantified on their arrays would be the glycosylated forms.

Another benefit of looking at only the glycoprotein fraction of their samples is that the researchers are able to load more sample. Because the array captures only a portion of the sample proteome, "you aren't limited to the [typical] capacity of the RPPA platform," Tao said. This means researchers are able to start with a larger sample volume than is possible with a conventional RPPA setup, which, in turn, means they are able to measure lower-abundance proteins.

The platform also benefits from an unanticipated boost in sensitivity due to the orientation of the molecules on the array. Often, Tao said, immobilizing a molecule on a bead or array surface can obscure portions of the epitope and therefore worsen antibody sensitivity. In the case of the polyGPA platform, the opposite happened — the array pulled down the target proteins by their glycosylations, leaving their epitopes more exposed to antibody detection and thereby improving the assay's sensitivity. In the JACS study, the researchers observed a roughly 10-fold increase in signal compared to conventional RPPA measurements when analyzing the α-1-acid glycoprotein (AGP) standard.

Using the polyGPA platform to measure endogenous AGP in human plasma, they likewise found it significantly more sensitive than a conventional RPPA assay.

Tao and his colleagues then used the platform to measure potential bladder cancer biomarkers in urine. Looking at a cohort of eight bladder cancer patients and five healthy controls, the researchers set out to validate three candidate markers, the proteins A2M, C4B, and ITGAM. While the lower-abundance C4B and ITGAM proteins were scarcely detectable by a conventional RPPA assay, they were easily measured using the polyGPA approach. Additionally, the authors noted, the polyGPA measurements showed that C4B was significantly elevated in bladder cancer patients, suggesting that C4B glycosylation could have potential as a marker for the disease.

One limitation of the polyGPA platform is its inability to distinguish between changes in glycoprotein levels due to changes in the overall expression of a target protein versus changes in the glycosylation levels of that protein. However, this can be determined by combining the method with other approaches, such as mass spec.

Calling the JACS study a proof-of-principle, Tao said that he and his colleagues are now working to establish that the polyGPA platform's functionalized surface is stable and can be manufactured reproducibly. The surface consists of a nitrocellulose membrane, as is commonly used in standard RPPA assays, that is functionalized with globular hydroxyaminodendrimers.

He added that to perform the assay in a more high throughput manner, the researchers would need to move from the manual spotting they used for the initial study to an automated arrayer, as is commonly used for large-scale RPPA experiments.

Tao said that in addition to continuing their urine-based work on bladder cancer samples, he and his colleagues are also using the platform for research into breast cancer biomarkers in plasma. They are collaborating with clinicians at the Indiana University School of Medicine on this work, he said.

He is also working to commercialize the method through Tymora, where he is chief scientific officer. Anton Iliuk, Tymora's president and chief technology officer, is a co-author on the JACS paper. The company's main products are research tools for phosphoproteomics work, though Tao said that Tymora has recently begun to consider potential clinical applications for its technology.