SARS-CoV-2's Spike protein is a multifunctional molecular machine that mediates the virus' entry into host cells. As such, a thorough understanding of the protein's biology, its structure, function and evolution, is critical to the development of therapeutics and preventives – such as vaccines – for eliminating the virus.
Dr Rod Chalk, head of mass spectrometry at the new Centre for Medicines Discovery at Oxford University (previously a part of the Structural Genomics Consortium) has developed a novel mass spectrometry-based method for characterizing glycans from the Spike protein. It can be executed using any liquid chromatography (LC) mass spectrometer, it does not require specialist knowledge of glycan analysis, and it takes advantage of the differential resolving power of a reversed phase high performance liquid chromatography (HPLC). Chalk and colleagues have created a database to share the data obtained through adopting this method and encourage its use by academics and industry.
Technology Networks spoke with Chalk to learn more about how the method works and its applications both in SARS-CoV-2 research and other infectious diseases. Chalk also discussed the realities of working on the SARS-CoV-2 virus in the current global pandemic.
Molly Campbell (MC): Can you talk to us about the "typical" research that takes place at the Structural Genomics Consortium (SGC) at Oxford University, and how this positioned you to study the SARS-CoV-2 virus?
Rod Chalk (RC): In August, the SGC became The Centre for Medicines Discovery. This explains what we do more succinctly. We identify proteins which are responsible for disease, clone and express them, then scale-up and purify them for structural analysis by X-ray crystallography or cryo-EM. Once we have obtained the structure, we then use a wide range of screening techniques to identify potential drugs which bind to it. All of this information, the methods and the reagents that we have made are placed in the public domain to be accessed by drug companies or other academic groups. Our aim is to accelerate the search for new drugs for cancer, Alzheimer’s disease, heart disease, etc. Because we have over 17 years’ experience of producing literally thousands of high-quality proteins, we are the “go to” lab for protein production and we're the natural choice for SARS-CoV-2 proteins.
Karen Steward (KS): Please tell us about the mass-spectrometry-based method you have been developing to improve the characterization of the SARS-CoV-2 spike protein.
RC: Our aim is to be able to identify, localize and quantify the myriad glycans which complicate the structure of the SARS-CoV2 Spike protein. Our method relies on generating short peptides bearing a single glycan, then identifying both the glycan and the peptide. To identify the glycan, we use accurate mass and the fact that all glycans from the same peptide share the same retention time by reversed phase HPLC. To identify the peptide, we use pseudo MS3 fragmentation. All this information is collated together in a database which now contains the accurate mass, retention time and structure for 406 Spike glycopeptides. This database paves the way for a much faster and simpler method which we call Mass-Retention Time Fingerprinting (MRTF). This will work in any mass spectrometry (MS) lab and does not required specialist knowledge of glycobiology. Users simply perform an overnight enzyme digest, a 60-minute liquid chromatography-mass spectrometry (LC-MS) run and then search their results against the MRTF database to derive the position, structure and relative intensity for each Spike glycan.
KS: Did you come up against any particular challenges in the development process and how did you overcome them?
RC: Yes. First, we found that trypsin generates peptides with more than one glycosylation site. Elastase is a much less popular choice, but we found it cuts where we needed it to and generates shorter peptides which are easier to work with. Next, we found that despite information in the literature, elastase cleavage is not 100% specific; however, we found that this did not actually hamper our method. Finally, we were unable to find a chromatography matrix which will separate all glycopeptides. We found reversed phase separates peptides well, but not with different glycans attached. We actually were able to make use of this, because if we found different glycans with the same retention time, we could use this to identify the peptide and hence locate the glycan on Spike.
KS: How can the data generated be utilized by researchers working towards an effective SARS-CoV-2 vaccine?
RC: Anyone expressing Spike needs to know that the surface glycans do not change significantly from batch to batch or from lab to lab. This may determine whether endogenous antibodies against Spike, or those engineered against Spike, will recognize it or not. In addition, the presence or absence of glycans will change the physical properties of Spike (molecular weight, solubility, charge, etc.)
MC: What other applications exist for this method outside of SARS-CoV-2 research?
RC: We developed this method specifically for SARS-CoV-2 Spike. However, heavy and complex glycosylation for the purpose of immune invasion is common to other viral pathogens such as Influenza, Ebola or HIV which has even more glycosylation sites than Spike. The basic strategy of building a mass-retention time database and then using it for rapid glycan fingerprinting could equally be applied to these and other proteins. Moreover, alternative glycan characterization methods have significant disadvantages. Released glycan analysis gives no positional information, fragmentation-based strategies require expensive equipment and considerable expertise and LC-MS strategies based on accurate mass are prone to high numbers of false positives. Our approach of using both accurate mass and retention time enables both rapid and confident glycan assignment.
MC: How has the COVID-19 global pandemic impacted your research, and how does it feel to be contributing to the ongoing efforts to develop a safe and effective vaccine?
RC: The COVID-19 epidemic has impacted all aspects of our research, and for several months all other research activities were halted. My colleagues and I volunteered to work on the coronavirus, and we have all learnt new science and new ways of working. Scientists have been willing to work incredibly hard and we have received many offers of help. Ultimately, everyone understands that scientists of all disciplines are key to defeating this threat, and we will eventually succeed with a successful vaccine, a drug treatment, a rapid diagnostic test or all three.
Dr Rod Chalk was speaking with Molly Campbell and Dr Karen Steward, Science Writers for Technology Networks.