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Dublin Scientists Team with Agilent to Develop 2D LC Separation Platform for Glycan Research


This story originally ran on July 8.

By Adam Bonislawski

Scientists at Dublin's National Institute for Bioprocessing Research and Training are partnering with Agilent Technologies to develop a two-dimensional LC separation platform for glycan research.

The platform will be based on Agilent's HPLC-chip technology and will enable high-throughput, nanoscale analysis of N-glycosylated proteins, Pauline Rudd, NIBRT professor of glycobiology at University College, Dublin, told ProteoMonitor.

Last week Agilent announced that it had presented Rudd, who is heading up NIBRT's portion of the collaboration, with its first Agilent Thought Leader Award in recognition of her work studying protein glycosylation. Over the last decade, Rudd's group has developed an HPLC-based platform for high-throughput N-glycan analysis and a glycome database called GlycoBase that contains elution time data for more than 400 glycan structures.

Her group is now working to develop an LC platform featuring two-dimensional separation for glycan analysis that will be adapted for use in Agilent's HPLC-chip system. Both Rudd's current platform and Agilent's existing glycan HPLC-chip feature only one dimension of separation.

"We want to combine two high-resolution phases for optimized two-dimensional separation," Rudi Grimm, director of science and technology for Agilent's life science group, told ProteoMonitor. "[Two-dimensional separation] is very typical in proteomics work, but to my knowledge it's never really been applied to the separation of glycans."

Diseases including rheumatoid arthritis and cancer have been shown to involve changes in glycosylation, suggesting that glycoproteins could prove useful as biomarkers. The structural complexity of these proteins makes their identification and quantitation difficult, however.

"A lot of biology really is happening at the post-translational modification level, and we understand more and more that changes in glycosylation very often lead to changes in disease and disease progression," Grimm said. "There is an increasing demand for having optimized systems for glycan analysis."

"A glycoprotein has one amino acid sequence, but it can have any number of different sugars associated with it," Rudd said. "And then it's also complicated because sugars aren't linear structures. A sugar ring has six carbons and those carbons can all link to other carbons in monosaccharides. So you get these complex branched structures, which means you can't just do straightforward mass spec to get the composition because that's not going to tell you which monosaccharides and how those monosaccharides are linked together."

To address these issues, Rudd and other researchers from the NIBRT, the University of Oxford, and Boston's Brigham and Women's Hospital developed the high-throughput HPLC-based platform currently in use in her lab. Described in a paper published in the May 2008 issue of Analytical Biochemistry, the automated, 96-well plate platform uses the GlycoBase database to assign structures to serum N-glycans based on HPLC profiling and exoglycosidase sequencing.

"We wanted to have a quantitative technology where you could take a student and within a week have them getting data," Rudd said. "If you want to know the ins and outs of enzyme digest, it's all in the database, but in order to actually do your data interpretation you don't need to know because the computer knows and takes into account enzyme specificities and all these arcane kinds of information."

Rudd added that she set out a decade ago to develop "something that anybody with basic experimental skills could use – and I think we've pretty much achieved it."

This May, Waters announced a partnership with the NIBRT to develop the institute's glycan database and assays for use with the company's UPLC technology. Scheduled to launch in 2011, the platform will be used with Water's UPLC Glycan Solution and will provide pharmaceutical companies with a tool for confirming the structures of biopharmaceuticals, many of which are glycosylated proteins (PM 05/07/10).

The UPLC platform also marks a step toward Rudd's goal of miniaturizing the NIBRT system.

"[UPLC] is much smaller, you need less material, you need less solvent, and it's faster," she said.

Via the collaboration with Agilent, Rudd plans to shrink the platform even further.

"What we want to do is take it down to a lower scale. We want to make it nanoscale," Rudd said. "We'd rather not have a big HPLC machine and lots of buffer. Agilent has very sophisticated chip technologies out there, so we want to work with their nanoscale technologies to miniaturize what we're doing."

"This is ultimately very important because in so many applications you're working with very limited amounts of sample," Agilent's Grimm said. "That's why you really need to have micro- and nano-based separation systems."

The HPLC-chip platform will also differ from NIBRT's current platform in that it won't use glycan derivatization or fluorescent labeling for detection, Grimm said.

"I'm interested only in methods that avoid any kind of derivatization process," he said. "Whenever you derivatize any component these reactions are never 100 percent – they take some time, you always need to remove the excess of the derivatization agent, and then in that procedure you always selectively enrich or remove some of the glycan components."

In addition to working with Rudd to develop separation media for incorporation into the planned two-dimensional HPLC-chip, Agilent will be collaborating with her on a new database for use with the chip, Grimm said.

"We'll set up a two-dimensional database, as well," he said. "That will be a completely new database. It's part of setting up this new multidimensional workflow."

The project is slated to launch within the next several weeks, Grimm said, with the goal being to have a prototype of the two-dimensional glycan HPLC-chip available within 12 months. According to the terms of the collaboration, technology developed by Rudd's lab during the project will be the intellectual property of the NIBRT.

"If we find commercial interest, we will try to get exclusive rights," Grimm said.

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