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Holland Group
Research Objectives


Microscale Processing to Rapidly Assay Protein, Antibody-Based Therapeutics.

The nanogel separations developed in the Holland Group separate oligosaccharides with unprecedented efficiency, using reagents <$1, and are completed in minutes. This has been demonstrated with the separation of branched glycan isomers by monomer sequence that differ only in the position of a single monomer linkage (b1-4 vs b1-3). These separations can quickly resolve complex glycan samples (i.e. efficiency of 640,000 theoretical plates). Upon discovering that the nanogel stabilizes the proteins better than aqueous solutions, the technology was patented. Small zones of nanogels that contain exoglycosidase enzymes, a class of enzyme which cleaves specific saccharide monomers at the terminal glycan structure, are patterned in the capillary to sequence these structures. When the enzyme specificity matches the terminal monomer, the substrate is cleaved and this cleavage is observed as a migration shift in the separation that follows, providing structural information without the need for biological standards or mass spectrometry.

Chair conformations of L-Fucose, D-Galactose, N-Acetyl-D-glucosamine, and D-Mannose alongside graphic of how they might be attached to a protein via glycosylation. Sample data from N-glycan sequencing with capillary electrophoresis included.

Capillary Electrophoresis-Vibrating Sharp Edge Spray Ionization.

Capillary electrophoresis separation is standard technology used widely for the analyses of proteins, DNA, drugs, and small molecules, but capillary electrophoresis coupled to mass spectrometry has not achieved this widespread implementation.  Capillary electrophoresis is a prominent technique for bioanalyses as a method of voltage-free coupling, and this technique is integrated with mass spectrometry within the research of the Holland Group.  This has led to our recent efforts in acoustically driven droplet formation as an innovative interfacing strategy to overcome some of these barriers. In this new design we perform electrophoresis with the detection end of the capillary in open air and focus energy directly at the liquid solution exiting the capillary at flow rates as low as 100 nanoliters per minute. 

Capillary Electrophoresis-VSSI-Mass Spectrometer showing how VSSI probe is attached to ground electrode. Details that process is: <$1 per probe $200 for interface; Low Flow (70-200 nL/min); and requires No voltage decoupling

Enabling Technology to Screen and Quantify Sialylated Structures for Activity Against Viral Enzymes and Receptors.

Both the economic and disease burden of viral infections in the United States are high, costing $11 billion for influenza in 2018 with 800,000 hospitalizations. Among the different biochemical targets, glycosylation is a powerful post-translational modification that plays a pivotal role in many viral infections. Receptor binding frequently involves sialic acid residues on cell surfaces and the release of virions replicated inside of a host cell is often enhanced by viral enzymes that cleave sialic acids on the cell surface, subsequently accelerating the infection of other cells. While this makes sialylation a target to intercept viral infections, the quantification and structural identification of sialic acids remains a challenge with current assays. This is because sialic acids, which have limited reference standards, are labile and easily degraded. The heterogeneity of sialic acid linkage isomers is further complicated by branched or modified sialylated compounds, leading to structurally similar and labile molecules. A new approach to elucidate enzymes and receptor interaction is introduced through the use of patterned nanogels in capillary electrophoresis. With this new strategy the sialic acid structures that interact strongly with enzymes or receptors are identified and screened in a high throughput manner. 

  Neuraminidase substrate screening, shows separation of enzyme and graphs time vs substrate/product with and without enzyme and inhibitor present. Michaelis-Menten curve for neuraminidase included, with a greater KM and lower rate with the inhibitor.