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Chasing a Challenging Molecule: Innovative Tools for Carbohydrate Analysis Open Doors for Research

Glycobiology is at a pivotal point, driven by urgent needs for carbohydrate structure analysis that can advance initiatives such as development and management of a COVID-19 vaccine. The latest special issue from SLAS Technology, Carbohydrate Structure Analysis: Methods and Applications, reveals new techniques to examine these intricate molecules.

Carbohydrates touch every aspect of life from plants, bacteria and animals, to medicines, makeup and food. Yet the diversity that makes them so compelling also makes them complicated to study, says Parastoo Azadi, Ph.D., technical director for Analytical Services and Training at the Complex Carbohydrate Research Center (CCRC), an organization dedicated to the interdisciplinary and equipment-intensive nature of carbohydrate science located at University of Georgia (Athens, GA, USA).

“Historically, carbohydrates are complex and heterogenous when compared to other biomolecules such as proteins. In order to elucidate their structure, we need specific, targeted tools beyond what we already have available, and that’s what this special issue of SLAS Technology addresses,” says Azadi, who serves as a co-guest editor for the special issue along with her CCRC colleague Christian Heiss, Ph.D., a senior research scientist.

Azadi and Heiss applaud the special issue authors who develop methods in spite of a spectrum of problems including the scarcity of standards, the presence of non-carbohydrate substituents and the difficulty of automating existing protocols for high-throughput analysis, to name a few. In their work at CCRC, the researchers face the same questions on a daily basis and hope this collection of research reveals room for growth in the field of glycobiology, says Heiss.

“We try to stay abreast of new developments and that was our motivation for developing this issue of SLAS Technology,” Heiss comments. “It’s exciting that scientists meet carbohydrate analysis challenges in such creative ways.”

Azadi agrees: “A number of these articles – such as one about glycomic analysis – have been developed for high-throughput analysis (HTA), something not seen much in this field. HTA provides an opportunity for glycobiology to be on par with proteomics and genomics analysis.”

Growth in glycobiology comes at a crucial time, she continues. "In the midst of this pandemic, scientists studying COVID-19 could benefit from the tools showcased in this special issue because a vaccine being developed may be based on the spike glycoprotein on the surface of the virus. These tools are necessary to make certain there is consistency in glycosylation structures within the developing vaccine, and moving forward, we want to make certain that subsequent batch analysis can be looked at as a whole. Nuclear magnetic resonance (NMR) and other techniques will be useful in monitoring the vaccine for contamination,” Azadi explains.

CCRC’s studies into SARS-CoV-2 have been plentiful during the crisis. As they examined the spike glycoprotein on the surface of the virus, Azadi and Heiss characterized the glycans on the spike, which is made of two subunits (S1 and S2). “There may not be a whole glycoprotein used for the vaccine but rather only the region of the S1 subunit that binds to the human host cell," Azadi explains.

Azadi and Heiss anticipate that the tools featured in the special issue will make such research easier and faster. They hope to capture a broader audience of scientists, even those not involved in glycoconjugate analysis. “Protein chemists, microbiologists, immunologists – people who may not be familiar with carbohydrate analysis can implement these tools in their labs,” Azadi continues. “They already have some of the instrumentation; they just don’t use it for carbohydrates.”

Breaking Down Monosaccharide Analysis

To offer a complete picture of the tools available, the co-editors conceptually divided the special issue into three distinct parts – monosaccharides, oligosaccharides and glycan-protein interactions. “This allows us to present a holistic view of the array of techniques,” Azadi explains.

In the section highlighting monosaccharide analysis, Heiss calls attention to an article by Jeffrey S. Rohrer, Ph.D., of Thermo Fisher Scientific (Sunnyvale, CA, USA). In “Vaccine Quality Ensured by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection,” an industry-standard chromatography technique is used to accurately and quantitatively determine the monosaccharides that are present in a sample. “The method even detects oligosaccharides,” Heiss says. “The paper gives an overview of how this technique has been used in the pharmaceutical industry in regard to carbohydrate vaccines.” 

Heiss mentions two articles in the section discussing methylation in plant polysaccharides. “No one knows why these substituents are there, and it is difficult to determine exactly where they are,” explains Heiss. “These papers elucidate new tools to determine the location and function of the methyl groups.”

In "Locating Methyl-Etherified and Methyl-Esterified Uronic Acids in the Plant Cell Wall Pectic Polysaccharide Rhamnogalacturonan II," author Malcolm A. O’Neill, Ph.D., and colleagues from the CCRC and the National Renewable Energy Laboratory (Golden, CO, USA) describe a new procedure using aqueous sodium borodeuteride (NaBD4) reduction of rhamnogalacturonan II (RG-II) to identify the methyl esterification status of backbone galacturonic acids (GalAs). The team’s data suggest that up to two different GalAs are esterified in the RG-II backbone.

Another article focusing on methylation, “Analytical Techniques for Determining the Role of Domain of Unknown Function 579 Proteins in the Synthesis of O-Methylated Plant Polysaccharides,” describes methods for the analysis of the 4-O-methyl glucuronic acid moieties that are present in sidechains of arabinogalactan proteins. These methods are then applied toward the analysis of loss-of-function mutants of two DUF579 family members that lack this modification in muro. Peter J. Smith, Ph.D., and fellow authors from CCRC, the University of Georgia’s Department of Biochemistry and Molecular Biology and the Center for Bioenergy Innovation, Oak Ridge National Laboratory (Oak Ridge, TN, USA), also present a procedure to assay DUF579 family members for enzymatic activity in vitro using acceptor oligosaccharides prepared from xylan of loss-of-function mutants, an approach that facilitates the characterization of enzymes that modify glycosyl residues during cell wall synthesis and the structures that they generate.

A final article in the monosaccharide section describes a derivatization method. In "Flow Chemistry System for Carbohydrate Analysis by Rapid Labeling of Saccharides after Glycan Hydrolysis," author Wei-Ting Hung, Ph.D., and colleagues from The Genomics Research Center (Taipei, Taiwan) and Department of Chemistry, National Taiwan University (Taipei, Taiwan), demonstrate the utilization of a flow chemistry system for continuous glycan hydrolysis and saccharide labeling to assist with the existing methods in glycan structural analysis.

“Analyzing monosaccharides is challenging because of the difficulty of separating them,” explains Heiss. “They are so similar to each other and do not get retained well in a lot of separation columns. They also don’t have a chromophore, making it difficult to detect them. The method that Hung and colleagues developed addresses all these things at once.”

The team starts with a naphthalene-2,3-diamine (NADA) and makes monosaccharide fluorescent images that “are pure and general so you can analyze all neutral sugars,” Heiss continues. “Also, they have been able to do this in a flow reactor, which means it can be done automatically, enabling high-throughput analysis without a lot of operator input. Traditionally each analysis takes many hours or even days to complete. If you want to analyze a lot of samples, it is necessary to have high-thoughput methods.”

Quick and Easy Glycomics

The glycomics section of the special issue features an article from CCRC scientists, "Simplifying Glycan Profiling through a High-Throughput Micropermethylation Strategy.” Author Asif Shajahan, Ph.D., and colleagues explore a high-throughput method for permethylation of released glycans using instruments commonly found in most laboratories. The team reports the wide potential of micropermethylation-based, high-throughput structural analysis of glycans from various sources, including human plasma, mammalian cells and purified glycoproteins, through an automated tandem electrospray ionization–mass spectrometry (ESI-MSn) platform.

“The permethylation helps to perform the mass spectrometry analysis in a more sensitive and informative way,” says Heiss, who along with Azadi, was involved in the research. “The article also includes how the standard set up for this particular mass spec can be modified to make the method high-throughput without investing a lot of extra money.”

A slightly different approach for achieving high-throughput is revealed in “High-Throughput Analysis of Fluorescently Labeled N-Glycans Derived from Biotherapeutics Using an Automated LC-MS-Based Solution.” Author Ximo Zhang, Ph.D., and fellow authors from the Waters Corp. (Milford, MA, USA), apply the optimized method to 48 released glycan samples derived from six batches of infliximab to mimic comparability testing encountered in the development of biopharmaceuticals. The data acquisition and analysis of the 48 samples were completed within six hours, representing a 90 percent improvement in throughput compared with conventional liquid chromatography–fluorescence–mass spectrometry-based methods. 

“Their method reduces the analysis time by running on a shorter column at a high flow rate,” Heiss says. “The disadvantage of that is that the peaks started running together. However, because you are using mass spectrometry for the detection method, you can still tease apart the different compounds that come out.”

The final glycomics article, “O-Benzylhydroxylamine (BHA) as a Cleavable Tag for Isolation and Purification of Reducing Glycans,” explores the use of a cleavable tag for the regeneration of free reducing glycans without affecting the structural integrity of glycans. Author Ying Zhang, Ph.D., and fellow authors from the Department of Biochemistry, Emory Comprehensive Glycomics Core, Emory University School of Medicine (Atlanta, GA, USA), develop a method for oxidative release of natural glycans (ORNG) for large-scale release of N-glycans as free reducing glycans.

“The BHA tag gives glycans chromophores so that they can be detected,” Heiss explains. “In the past, putting on a chromophore destroyed the original glycan; you couldn’t use it for anything else because it had been modified.” The tag can be easily removed by palladium-on-carbon (Pd/C)-catalyzed hydrogenation to efficiently regenerate free-reducing glycans with little effect on glycan structures.

Protection and Detection of Glycan-Protein Interactions

The section on glycan-protein interaction begins with a review from William P. Vignovich, Ph.D., and Vitor H. Pomin, Ph.D., of the BioMolecular Sciences Department and the Research Institute of Pharmaceutical Sciences, respectively, of the School of Pharmacy at the University of Mississippi (Oxford, MS, USA). "Saturation Transfer Difference in Characterization of Glycosaminoglycan-Protein Interactions" focuses on the latest achievements using saturation transfer differences (STD) of NMR spectroscopy to study the interactions of proteins with glycosaminoglycans (GAGs), a group of sulfated polysaccharides that play pivotal roles in various biological and pathological processes. STD specifically determines which parts of ligands interact with the protein, which is significant for the study of structure-activity relationships.

“The method detects when a small molecule binds to a large molecule – like a protein. Determining what parts of the small molecule interact with the protein is important to understand the binding mechanism and how to change the small molecule so that it binds better,” says Heiss.

The last article explores glycan-protein interaction in a different way. In “Preparation and Application of Nanosensor in Safeguarding Heparin Supply Chain,” author Yiling Bi, Ph.D., and fellow authors from the Departments of Biology, Bioengineering and Medicinal Chemistry, University of Utah (Salt Lake City, UT, USA), develop a sensitive method to detect oversulfated chondroitin sulfate (OSCS) in heparin lots using a nanosensor, gold-nanoparticle-heparin-dye conjugate that can detect as low as 1 × 10-9% (w/w) OSCS.

“In 2008, several people died from contaminated heparin. This team’s quick, sensitive detection method can prevent that from happening again,” says Heiss. “When you look at the difficulties associated with carbohydrate analysis, it’s easy to think that they’re impossible to overcome, but they’re not.”

Azadi agrees. “It’s intriguing how scientists have come up with such diverse methods and techniques to be able to dig in and look at these challenging molecules,” she concludes. "Sharing these highlights and developing new high-throughput methods will expand the audience for carbohydrate analysis because of its effects in research. I think researchers can see that these tools are easy to use and implement in most laboratories.”

Learn More

The SLAS Technology special issue is available now at SLAS Technology Online for SLAS members, SLAS Technology subscribers and pay-per-view readers. Free public access becomes available one year after final (print) publication.

Featured Podcast: Carbohydrate Structure Analysis: Methods and Applications

Join Parastoo Azadi, Ph.D., and Christian Heiss, Ph.D., of the University of Georgia (Athens, GA, USA), as they discuss the SLAS Technology Special Issue on Carbohydrate Structure Analysis: Methods and Applications in a 2020 SLAS Technology podcast.

July 26, 2020