By Lance Wells, Co-Director of the Thermo Fisher appointed Center of Excellence in Glycoproteomics at the CCRC at the University of Georgia.
As we race to combat the COVID-19 pandemic, time is of the essence. Technological advancements are required to understand the behaviour of the COVID molecule to develop vaccines that will prevent the virus, and arrive at a treatment we can be confident will have the highest level of effectiveness.
The Complex Carbohydrate Research Center (CCRC) at the University of Georgia has had access to new laboratory instrumentation technology. Not only does this potentially enable us to do better, more thorough analysis of the COVID virus, it may usher in a new age in pharmaceutical development, with more comprehensive studies leading to more effective treatments.
CCRC works in various disciplines – biomedical, plant and microbial glycosciences, synthetic and analytical chemistry – with scientists around the world. We are involved in coronavirus research, to better understand SARS CoV2, the infectious agent for COVID-191. The glycosylated spike protein on the coronavirus molecule’s surface is a glycoprotein, responsible for promoting entry of the virus into host cells; it is also the target of neutralising therapeutics. By understanding the properties of glycoproteins we gain insight into how to block the virus from binding to host receptors, and mechanistic information to develop effective vaccines and therapeutics.
Our research has required liquid chromatography and mass spectrometry (LC-MS), a combination that has limitations – primarily because of the front end liquid chromatography component. The technology is complicated to use and suffers from inadequate resolution for the speed that coronavirus research demands.
CCRC was recently introduced to High Resolution Ion Mobility. The technology is intended for fast, efficient, high-resolution instrumentation for biomarker discovery, diagnostics and therapeutic development. This next generation ion mobility technology, based on ‘SLIM’ technology (Structures for Lossless Ion Manipulation), has the potential to dramatically improve the throughput of samples for analysis in research labs. Its superior resolution allows SLIM technology to detect what conventional instrumentation might leave unnoticed.
Easier to use, better separations, reproducible findings
SLIM technology is less challenging and less complicated to use than LC. Low flow rate LC requires a level of expertise that has not kept up with the advances of other instrumentation, like mass spectrometry. Twenty years ago, mass spectrometry demanded a high level of expertise. Today, if the instrument is tuned, calibrated, and working efficiently, I can train a graduate student in two days. Often they may be just as competent as my postdoctoral associates who may have been working with the technology for years.
The bottleneck usually comes in creating LC separations. Analysing glycans released from COVID-19 requires three to five hours of separation by LC for a single clinical isolate. We are now looking at some 40 different clinical isolate variants for COVID-19 expressed in a variety of systems. With the time required for conventional separations, we may be able to run only as many as three per day, which can hinder research. (That’s true for any clinical sample. Being able to turn two or three samples a day is just not effective.)
Our first SLIM technology experience was analysis of a complex glycan sample that we had analysed over a four-hour LC gradient. With SLIM, we were able to complete an analysis in a two minute run, and we were able to see all the glycans that we saw in four hours of LC separation. That is a 120 fold gain in analysis time, and when time is of the essence – as in COVID research – that’s not only impressive, it’s critical.
Another benefit of SLIM technology is reproducibility of findings. We’ve found the reproducibility with SLIM is as good and probably better than LC.
This improved reproducibility is because separations in SLIM are based on a physical property (the collision cross section) of that molecule, which never changes. No matter where or by whom a sample may be run, it behaves the same way.
That is not true of LC. Factors including temperature fluctuation and the age of the column make LC reproducibility challenging. Laboratories typically use standards to renormalise the gradients in LC separations. With SLIM technology, the immutable physical property of the molecule assures more consistent results. If we run a sample at CCRC, another is run in Europe, and a third is run in Australia, all will get the same data.
Separations on printed circuit boards
Imagine SLIM technology as two parallel, mirror-image printed circuit boards (PCBs) close together, but not touching. Voltages applied to the PCBs create an electric field-based conduit for the ions to traverse in the gas phase without touching the PCB surfaces. Separation is achieved by applying a wave-like voltage to the ions to propel them forward, traveling the path length created on the PCB. Smaller, more compact ions move forward faster than larger ions, and separation is achieved as the ions move forward along the separation path length.
With ion mobility, resolution, or the degree of separation, is predicated on path length: The longer the paths, the better the separation. With SLIM, two 30 cm x 30 cm circuit boards allow for a 13 metre (40 foot) path length in a device the size of a laptop. This is a very long path despite its relatively small footprint. Ions follow serpentine paths and turn corners between the printed circuit boards, resulting in very high resolution separations, separating and revealing molecules other instruments fail to achieve.
Unlike LC, SLIM separations occur in the gas phase. Ionized molecules are separated based on size, charge and shape, which happens more quickly in this phase. Analytes with the same molecular mass and chemical formula are separated by their size, shape and structure, rather than their chemical affinity with a particular buffer or solid phase support, as with LC separations. SLIM also can accumulate ions for ease of analysis and selectively switch them as a group to other locations, without any losses.
With this technology, we can achieve separations of isomeric molecules (molecules of the same mass and same chemical formula). Samples of varying analyte classes can be run back-to-back without component change-out. Method development is faster, and instrument uptime should generally be greater.
Seeing what other technologies leave unseen
These technological advantages are positioned to enable us to perform very high throughput analysis of glycans and glycoproteins, which otherwise are very difficult to analyse. We may not even know what a ‘normal’ range may be in glycans from a human blood sample.
SLIM technology is proving to have a superior capability to separate and identify the most challenging clinically significant molecules – even many that may escape detection by LC-MS processes. Combined with software-driven methods, better instrument uptime, and reduced operator cost, SLIM performs analysis significantly faster than traditional methods.
Replacing LC with SLIM as the separation workhorse makes it easier to consider moving mass spectrometry into clinical settings. We were able to get much improved resolution while reducing the time required from four hours to two minutes. Where we might have been able to run four samples in a day, this instrumentation allows those same four samples to be run in 15 minutes – hundreds in a typical week.
Running hundreds of samples so quickly allows us tighter analysis to establish the baseline for a “normal” glycan range. Consider analysing 240 tissue samples to identify the frequency with which a particular glycan can be detected. Analysing 240 samples using LC can take half a year. Using SLIM technology, the analysis can be done in no more than two weeks. That allows us to gain greater insight into the behaviour of glycans in a range of illnesses beyond COVID.
The future of disease studies and treatment
I believe that SLIM technology may help move mass spectrometry into broader clinical applications. It has none of the challenges typically associated with LC. It does not require the same level of expertise. It is designed to be more of a “plug and play” type of instrument which a technician can be trained to use in less than a week, and begin analysing new samples every five to 10 minutes, with better resolution and consistently reproducible data.
When creating disease treatments, the time required is painful for researchers, but the true tragedy is the patient’s. Anything we can do on the research side to speed up analysis, to discover more effective treatments, we must consider.
Using conventional LC, the industry in general is not particularly good at finding specific biomarkers quickly and efficiently. That adds time to disease treatment discovery (and efficacy), because many studies can be argued to be underpowered.
Consider cancer treatments. We may only have the capacity to analyse 10 tumours, when we really need to look at 200 or more, but we are at the mercy of time. We may not be able to confidently identify sufficient appropriate biomarkers.
Lossless, high resolution, high throughput technology like SLIM has enormous potential. We can test markers more efficiently from the start, with much higher throughput, much higher numbers, and much higher resolution, which may translate to saving time on the backend. If we can design better studies where we analyse hundreds of samples, we will arrive at more effective disease treatments in the same amount of time.
Volume 22, Issue 1 – Winter 2020/21
About the author
Lance Wells is a Georgia Research Alliance Distinguished Investigator, Professor of Biochemistry and Molecular Biology, Director of Integrated Life Sciences, and co-director of the Thermo Fisher appointed Center of Excellence in Glycoproteomics at the CCRC at the University of Georgia.
References
1 Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human ACE2 Receptor.
