Custom HPLC-Chip enables new research in glycan expression

By Dayin Lin
Product Manager, Liquid Phase Analysis Division, LSG, Agilent Technologies, Inc.


	Customer Case Study: Mass Spectrometry Resource, Boston University School of Medicine, Boston, Massachusetts, U.S.A.

Progress in biomedical research often takes a quantum leap when the right instrumentation becomes available. Glycan expression is one of those examples. Structurally diverse glycans are found on mammalian cell surfaces and in extracellular matrices. They mediate cell-cell recognition and cell-cell and cell-matrix interactions, but the exact details are largely a mystery. An innovative Agilent HPLC-Chip has now solved many of the difficulties that were inherent with LC/MS characterization of these compounds.

Figure 1. Graduate student Greg Staples (left) and Professor Joseph Zaia (right) use this Agilent HPLC-Chip/MS system for leading-edge research on glycoconjugates.

Professor Joseph Zaia and his team at the Mass Spectrometry Resource within the Boston University School of Medicine develop LC/MS methods to study the range of glycans that are expressed under various cell conditions.[1],[2] While nanoflow LC/MS with negative ion electrospray is the method of choice for characterization and quantification of these diverse carbohydrates, researchers must overcome a number of challenges. We spoke with Professor Zaia about the impact the HPLC-Chip has made in his research.

Rapid setup and stable performance

Before the HPLC-Chip, the lab’s major problem resided in the interface between the LC and the MS. Achieving a stable spray in negative ion mode at nanoflow rates was difficult, so it took a long time just to set up an experiment. That all changed the first time they used the HPLC-Chip – a small device with microfluidic channels, HPLC column packings, an integral nanoelectrospray emitter, and electrical contacts for electrospray. Zaia commented, “We quickly realized that the HPLC-Chip was engineered to solve this problem, and that it was no longer going to be a struggle to acquire the data. The HPLC-Chip was an enabling technology for us.”


	“The HPLC-Chip was an enabling technology for us.”

Based on this initial success, Zaia’s group purchased an Agilent HPLC-Chip/MS system that included an Agilent 6520 Accurate-Mass Q-TOF LC/MS. At about the same time, they applied for and received a university grant from the Agilent Foundation, which began a collaborative effort that has introduced new capabilities to the scientific community.[3]

Figure 2. This HPLC-Chip incorporates a makeup flow, which helps to achieve stable electrospray when the LC gradient provides less than ideal conditions. (Click here to see this image larger.)

Makeup flow enables overnight runs

Zaia explained that his team uses hydrophilic interaction liquid chromatography (HILIC), which requires a custom chip with a special packing material. The gradient runs from high to low organic, which ordinarily makes it difficult to get a stable spray in negative mode at the end of the run. To solve this problem, Agilent collaborators provided an experimental makeup flow (MUF) HPLC-Chip (Figure 2), which allows Zaia’s team to add organic solvent post-column. They achieve a consistent spray over the entire gradient, which extends the range of glycans they can measure. And they no longer need to adjust source voltages during the run, so they can now perform overnight, unattended analyses.

Now possible: analysis of large sample sets

Professor Zaia spoke about the large-scale studies he can now conduct to examine differences in the temporal and spatial expression of glycosaminoglycans (GAGs). For this critical research, the LC/MS system must provide consistent, reliable data for triplicate runs of dozens of samples. “The system has to be stable for days to get through the set of samples we need to handle. Over the past year and half, we’ve been able to use the system for these large sample sets. Previously, we would not have had the instrument stability to address these research questions.”


	“Previously, we would not have had the instrument stability to address these research questions.”

A new way to sequence fragile GAGs

The team is excited about future plans to further characterize GAGs using LC/MS/MS. GAGs are particularly challenging because many are sulfated, which makes them quite labile. Zaia plans to use the makeup flow to introduce an additive that will stabilize the sulfate group so he can get useful backbone fragmentation without loss of side groups. He cites the continued collaboration with Agilent as a key to future success. “Agilent has been very supportive in working with us on this technology. We look forward to the move into online tandem MS to do more detailed characterization.”

The custom HPLC-Chip has given Zaia’s lab a research tool that enables new biomedical discoveries at Boston University. In addition to custom HPLC-Chips, Agilent offers standard HPLC-Chips for a number of applications; all are compatible with Agilent’s 6000 Series LC/MS systems. To learn more, see the Agilent product page, or talk with your Agilent Representative.

References

J. Zaia, “On-line separations combined with MS for analysis of glycosaminoglycans,” Mass Spectrom Rev. 28(2):254-72, 2009.
J. Zaia, “Mass spectrometry and the emerging field of glycomics,” Chem. Biol. 15(9):881-92, 2008.
G. O. Staples, M. J. Bowman, C. E. Costello, A. M. Hitchcock, J. M. Lau, N. Leymarie, C. Miller, H. Naimy, X. Shi, and J. Zaia, “A chip-based amide-HILIC LC/MS platform for glycosaminoglycan glycomics profiling,” Proteomics, 9(3):686-95, 2009.

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