High resolution mass spectrometry of respiratory viruses: beyond MALDI-ToF instruments for next generation viral typing, subtyping, variant and sub-variant identification.

In the wake of the SARS-CoV2 pandemic, a point has been reached to assess the limitations and strengths of the analytical responses to virus identification and characterisation. Mass spectrometry has played a growing role in this area for over two decades, and this review highlights the benefits of mass spectrometry (MS) over PCR-based methods together with advantages of high mass resolution, high mass accuracy strategies over conventional MALDI-ToF and ESI-MS/MS instrumentation. This review presents the development and application of high resolution mass spectrometry approaches to detect, characterise, type and subtype, and distinguish variants of the influenza and SARS-CoV-2 respiratory viruses. The detection limits for the identification of SARS-CoV2 virus variants in clinical specimens and the future uptake of high resolution instruments in clinical laboratories are discussed. The same high resolution mass data can be used to monitor viral evolution and follow evolutionary trajectories.

Resolving omicron sub-variants of SARS CoV-2 coronavirus with MALDI mass spectrometry.

Mass mapping using high resolution mass spectrometry has been applied to identify and rapidly distinguish the omicron sub-variants across the BA.1-BA.5 lineages. Lineage-specific protein mutations in the surface spike protein give rise to peptide biomarkers of unique mass that can be confidently and sensitively detected with high resolution mass spectrometry. Those that are most efficiently ionised and detected within the S1 subunit in recombinant forms facilitate their detection in clinical specimens containing other SARS-CoV2 viral proteins and contaminants. A study of five dozen omicron-positive specimens, using a selected ion monitoring approach, detected peptide biomarkers for strains of BA.1, BA.2.75 and BA.4 sub-variants in 23%, 42% and 28% of samples respectively, consistent with their reported levels in the local population. The virus was confidently assigned in over 93% of omicron positive specimens. The ease of detection of the BA.2.75 variant, in particular, is of vital importance given its rapid global spread in late 2022 due to several immune evasive mutations within the receptor-binding domain.

Charting and tracking the evolution of the SARS CoV-2 coronavirus variants of concern with protein mass spectrometry.

The evolution of the SARS-CoV2 coronavirus spike S-protein is studied using a mass spectrometry based protein phylogenetic approach. A study of a large dataset comprising sets of peptide masses derived from over 3000 proteins of the SARS-CoV2 virus shows that the approach is capable of resolving and correctly displaying the evolution of the major variants of concern. Using these numerical datasets, through a pairwise comparison of sets of proteolytic peptide masses for each protein, the tree is built without the need for the sequence data itself or any sequence alignment. In the same analysis, single point mutations are calculated from peptide mass differences of different protein sets and these are displayed at the branch nodes on the tree. The tree topology is found to be consistent with that generated using conventional sequence-based phylogenetics by a manual visualisation and using a tree comparison algorithm. The mass tree resolves major variants of the virus and displays non-synonymous mutations, calculated based on the mass data alone, on the tree that enable protein evolution to be charted and tracked along interconnected branches. Tracking the evolution of the SARS-CoV2 coronavirus S-protein is of particular importance given its role in the attachment of the virus to host cells ahead of viral replication.

Detection of SARS CoV-2 coronavirus omicron variant with mass spectrometry.

Mass mapping using high resolution mass spectrometry has been applied to identify and rapidly distinguish the omicron variant of the SARS-CoV2 coronavirus strains from other major variants of concern. Insertions, deletions and mutations within the surface spike protein result in associated mass differences in the mass maps that distinguish the variant from the originating strain and the preceding alpha, beta, gamma and delta variants of concern. The same mass map profiles can also be used to construct phylogenetic trees, without the need for protein (or gene) sequences or their alignment, in order to chart and study the origins of the variants, or any other strains. The speed and sensitivity of mass spectrometric analysis is demonstrated for a preliminary set of clinical specimens with comparable sample handling to that required in PCR based approaches.