4 February / March 2021
many different glycan structures can have the same mass, charge, and physical properties, but vary widely in function and recognition. Separation and identification of these complex isomeric materials can be exceedingly time-consuming and resource intensive.
The State of Glycan Analysis Workflows
Figure 2: MOBILion’s High Resolution Ion Mobility Instrument coupled to an Agilent Q-ToF.
$7 billion blockbuster CD20-targeting mAb, Rituxan (rituximab). Patients receiving Gazyva showed greater depth of remission and increased progression-free survival compared to those who received Rituxan [9].
Additionally, glycosylation variation in patients can correlate with treatment outcomes. In order to optimise treatments and clinical trial outcomes (e.g. patient stratification), it is critically important to have the ability to identify glycosylation profiles in a high throughput manner.
With respect to vaccine development, another example of the clinical impact of glycans includes the HIV-1 envelope proteins, which is richly decorated with glycan structures that help the virus evade recognition by the immune system. This so-called ‘glycan shield’ is an active area of research and serves as a potential target for vaccine development and broadly neutralising antibody production [10]. Similarly, the COVID-19 pandemic has brought attention to the role the SARS- CoV-2 spike protein glycosylation plays throughout viral infection and in therapeutic development [11,12]. Building on research into broadly neutralising antibodies against HIV [13], researchers are now also looking to apply a similar approach to coronaviruses [14].
The Challenge of Glycan Analysis
To leverage the potential of
glycoengineering and enhanced knowledge of glycosylation patterns, researchers are increasingly interested in utilising better
tools and methods for quickly and accurately determining glycan structures. However, despite impressive advances, glycan analysis still remains challenging, largely due the nature of glycans themselves.
Arguably the principal structural challenge is that glycans are one of the most structurally diverse biomolecule families [2]. Though most mammalian glycans are built from around nine monosaccharide units, these monomers can be attached to one another through many different glycosidic linkages, including in a branched fashion [15]. Furthermore, glycans may range from one monosaccharide to many. Collectively, the number of possible glycan structures quickly expands with each additional monomer and branch. It is estimated that for a hexasaccharide there are, in total, ~1012 possible glycan structures, though they may not all occur in nature [16].
In addition, glycan assembly is not template-driven like DNA, RNA, and protein biosynthesis. Instead, glycans are made through interlaced networks of glycosyltransferases acting in the endoplasmic reticulum and Golgi. As a result, different glycan structures can occur at the same glycosylation position, generating biomolecules that are identical except for the glycans they carry (i.e. glycoforms). The distributions of glycoforms for a given glycoconjugate are also subject to change as glycosyltransferase expression changes.
Importantly, many of the monosaccharides that make up glycans are isomers of one another - glucose, galactose, and mannose being prime examples. This means that
As described above, glycan structures are inherently complex and heterogeneous. The go-to technique for glycan analysis has long been mass spectrometry (MS) - it offers the resolving power and sensitivity needed to elucidate specific glycan structures and glycoforms isolated from biological materials [17,18]. However, to achieve meaningful resolution between highly similar glycans within the same sample, separation techniques are needed ahead of MS analysis.
On a practical level, LC separation of highly similar biomolecules - like many glycans are - often requires extended run times, creating a potential bottleneck in analytical workflows. Even with long run times, some glycans behave so similarly that LC coelution cannot be avoided, which complicates or even prevents full structural assignment [19].
So, while LC-MS remains the standard workflow for glycan analysis, there is a pressing need for faster techniques with better resolving power.
More recently, High Resolution Ion Mobility Mass Spectrometry (HRIM-MS) based on Structures for Lossless Ion Manipulation (SLIM) has emerged as a promising separation strategy in glycan analysis [20]. Unlike liquid chromatography, MOBILion’s HRIM system separates ionised molecules in the gas phase. The system uses printed circuit boards (PCBs), see Figure 1, held in a chamber maintained at constant pressure (2-4 torr). The PCBs have a series of radio frequency (RF), direct current (DC) and traveling wave electrodes printed on them that provide an ion conduit through which the analytes traverse along an exceptionally long serpentine path. The electric fields that propel the ions also prevent them from striking surfaces while moving, therefore preventing any losses along their way. Depending upon the speed of the traveling wave, ions either ‘surf’ and are not separated, or they undergo enough collisions with the gas molecules present that they roll over the traveling wave peaks, and separation occurs.
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