As the SARS-CoV-2 pandemic made landfall...

research groups embraced the challenging work of deciphering the mechanisms of spread and infection. The new coronavirus employed a pronounced spike protein on the virus surface, now proven to be the central mediator of infection through interaction with host cell receptors. Due to its potential role in immunogenicity, the spike protein has emerged as the premier protein towards which a growing number of vaccine candidates are targeted.

An interesting aspect of early work included discovery of the densely glycosylated, sugar-like molecules covering the protein surface. Questions immediately arose of whether the glycans were responsible for the solubility characteristics, protein folding, or perhaps part an immune avoidance strategy attributable to the protein. Uncovering the significance of this glycan coat could be essential in fighting the virus.

In a study exploring the subject, entitled “Site-specific glycan analysis of the SARS-CoV-2 spike”, LC-MS analysis techniques were used in mapping of the glycan-processing states across the spike complex. The work exemplified how SARS-CoV-2 spike glycans differ from typical glycan processing, with perhaps with major implications in drug and vaccine development.


Nano-LC Mass Spectrometry for Glycan Analysis

As a critical part of the spike protein glycan mapping project mentioned above, peptide samples were subjected to nano-LC ESI-MS using an Easy-nLC system coupled to a Fusion mass spectrometer (Thermo Fisher Scientific) using high energy collision-induced dissociation for peptide fragmentation.

Nano-flow LC-MS in general is regarded as a highly sensitive technique for analysis of low abundance or peptide-rich samples such as those in proteomics or protein profiling experiments. The technique is superior to normal flow LC for determination of peptide species, owing to the large dynamic ranges and low volume requirements of the nano-LC columns. In addition, systems can be coupled with online add-ons such as selective trapping columns and other tools to enable faster, more automated analysis.

The approach does come with certain conditions, including the need for sample consistency to avoid column interferences, the absence of matrix factors, and the requirements for systems with tight connections and minimal void volumes, to prevent diffusion and resolution loss. Somewhat limited compared with the robustness and higher-throughput of convention LC, nano-LC excels as a research technique for smaller-scale, detailed applications.


The spike protein glycan mapping project employed the Easy-nLC 1200 system from Thermo Fisher Scientific, the latest product to use the Easy-Spray technology which provides an integrated and temperature-controlled column-emitter design.

  • Use of a single Thermo Dionex nanoViper connection between the LC and the EASY-spray source addresses the most error prone and problematic issue with nano-LC, the dead volume issue, as discussed above.
  • From the nanoViper fitting connecting the sample line, the flow then enters the circular column housing, which allows columns of variable length to be coiled in a uniform configuration.
  • The housing includes an integrated column heater with an operating range 30-60 °C. An ZDV (zero dead volume) union connects the eluent to a metal cylinder, which provides high-voltage to the glass spray tip source. A built-in camera permits magnified view of the emitter source and spray.

The columns used in the spike protein glycan mapping study included an EasySpray PepMap RSLC C18 column (75 μm × 75 cm) used inline with a PepMap 100 C18 3 μM 75 μM x 2 cm trapping column connected upstream.

  • A range of columns are available with various chemistries, particle sizes ranging from 2-5 µm, diameters of 50-150 µm, and varying lengths to accommodate required peak loading capacities.

Beyond the SARS-CoV-2 glycan mapping project, many fields and applications are benefiting from new, state-of-the art glycan analysis technologies.


Agilent offers the AdvanceBio glycan mapping solutions including a collection of glycan analysis columns to suit various applications.

  • These AdvanceBio columns are designed and manufactured for fast, high resolution, reproducible performance using HILIC chromatography. 
  • A common medium used for glycan separations, Hydrophilic interaction liquid chromatography (HILIC) is a variant of normal phase liquid chromatography, in which the hydrophilic stationary phase is coupled with reversed-phase eluent solvents. Through liquid-liquid partitioning, analytes elute in order of increasing polarity, with overlapping yet distinct advantages to ion mobility or reversed phase separations.
  • Two main types of bead sizes are available with the AdvanceBio columns, 2.7 µm superficially porous, for high resolution and lower backpressure, and 1.8 µm for high resolution.

The AdvanceBio glycan mapping columns are part of the complete suite of glycan analysis solutions from Agilent.

  • Sample prep kits, N-glycan labels, standards, enzymes for structural characterization, and HILIC columns provide tools for every level of analysis, from intact glycoproteins to released N-glycans.

Waters offers innovative technology to support glycan analysis in biopharma applications.

  • The Acquity QDa Detector enables both optical and mass analysis techniques to be run inline in a single screening workflow.
  • This greatly improves flexibility and throughput in peptide biomarker screening, quality control procedures, and glycan analysis.
  • The ability to employ mass spec detection using a simple to connect plug and play format extends the use of Acquity systems significantly.
  • The dual detection arrangement allows mass spec monitoring of scouting experiments, with faster time to scale-up to feasibility and production.

The New RapiFluor-MS sample prep system from Waters enables released glycan analysis using both fluorescence and mass spec detection modes.

  • The new GU Scientific Glycan Library for Released N-Glycans facilitates characterization of glycan species, including high mannose, complex alpha-lined galactose pairs, and hybrid glycans.
  • Applications include monoclonal and therapeutic antibody analysis and quality control.

Mobilion Systems has unveiled a novel ion mobility product at the ASMS reboot virtual conference this year.

  • The patented SLIM device (Structures for Lossless Ion Manipulation) enables efficient separation, identification, and analysis of the most challenging molecules many other LC-MS systems fail to detect.
  • SLIM provides rapid, high resolution separations in the gas phase on printed circuit boards, effectively digitizing separations prior to MS analysis.
  • With a 13-meter serpentine ion path length in a component that is 14 x 18 inches, the new product surpases linear path techniques for unparalleled resolution and throughput.
  • SLIM can be integrated with LC-MS workflows and in some cases replace liquid chromatography to provide greater speed, efficiency, and ease of use.

Mobilion has recently partnered with Agilent to combine SLIM with Q-TOF mass spectrometry, to eliminate LC-MS bottlenecks and enable multi-dimensional analysis of new and relevant molecules.

  • As well, a recent collaboration with researchers at the Complex Carbohydrate Research Center (CCRC), University of Georgia, aims to detail the glycosylation microheterogeneity in the spike protein on the SARS-CoV-2.
  • Again, understanding the role of these glycans in receptor binding may be essential in determining effective COVID-19 treatments.

Summary

New technologies abound for many areas of research, biopharma development, and quality control. Areas including basic research into the SARS-CoV-2 spike protein glycan coat are contributing to the development of viable vaccines, drugs, and therapeutic antibodies -- all benefiting from these new glycan analysis and LC-MS solutions.


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