SEQUENCE-FREE PHYLOGENETICS WITH MASS SPECTROMETRY.

An alternative, more rapid, sequence-free approach to build phylogenetic trees has been conceived and implemented. Molecular phylogenetics has continued to mostly focus on improvement in tree construction based on gene sequence alignments. Here protein-based phylogenies are constructed using numerical data sets ("phylonumerics") representing the masses of peptide segments recorded in a mass mapping experiment. This truly sequence-free method requires no gene sequences, nor their alignment, to build the trees affording a considerable time and cost-saving to conventional phylogenetics methods. The approach also calculates single point amino acid mutations from a comparison of mass pairs from different maps in the data set and displays these at branch nodes across the tree together with their frequency. Studies of the consecutive, and near-consecutive, ancestral and descendant mutations across interconnected branches of a mass tree allow putative adaptive, epistatic, and compensatory mutations to be identified in order to investigate mechanisms associated with evolutionary processes and pathways. A side-by-side comparison of this sequence-free approach and conventional gene sequence phylogenetics is discussed.

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.

Distinguishing common SARS-CoV2 omicron and recombinant variants with high resolution mass spectrometry.

A selected ion monitoring (SIM) approach combined with high resolution mass spectrometry is employed to identify and distinguish common SARS-CoV2 omicron and recombinant variants in clinical specimens. Mutations within the receptor binding domain (RBD) within the surface spike protein of the virus result in a combination of peptide segments of unique sequence and mass that were monitored to detect BA.2.75 (including CH.1.1) and XBB (including 1.5) variants prevalent in the state's population in early 2023. SIM detection of pairs of peptides unique to each variant were confidently detected and differentiated in 57.3% of the specimens, with a further 10 or 17.5% (for a total of 74.8%) detected based on a single peptide biomarker. The BA.2.75 sub-variant was detected in 18.7%, while recombinant variants XBB and XBB.1.5 were detected in 13.3% and 25.3% of the specimens respectively, consistent with circulating levels in the population characterised by RT-PCR. Virus was detected in 75 SARS-CoV2 positive specimens by mass spectrometry down to the low or mid 104 copy level, with a single false positive and no false negative identified. This article is the first paper to characterise recombinant strains of the SARS-CoV2 virus by this, or any other, MS method.

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.

Analysis of bacterial biotyping datasets with a mass-based phylonumerics approach.

A mass tree, phylonumerics approach, is implemented for the first time with expressed protein mass data acquired in biotyping applications. It is shown, for two separate and diverse bacterial datasets, that the MassTree algorithm can be used to build phylogenetic trees in a single step that mirror those output by biotyping analysis software in the form of a main spectral profile (MSP) dendrogram or alike. Adapted for these applications to accommodate higher mass inputs and large mass error tolerances for pairwise matching, the mass tree algorithm and approach offers an alternative to commercial biotyping platforms by utilizing datasets acquired from any mass spectrometer without the need for specialized and expensive software.

Mass spectrometry analytical responses to the SARS-CoV2 coronavirus in review.

This article reviews the many and varied mass spectrometry based responses to the SARS-CoV2 coronavirus amidst a continuing global healthcare crisis. Although RT-PCR is the most prevalent molecular based surveillance approach, improvements in the detection sensitivities with mass spectrometry coupled to the rapid nature of analysis, the high molecular precision of measurements, opportunities for high sample throughput, and the potential for in-field testing, offer advantages for characterising the virus and studying the molecular pathways by which it infects host cells. The detection of biomarkers by MALDI-TOF mass spectrometry, studies of viral peptides using proteotyping strategies, targeted LC-MS analyses to identify abundant peptides in clinical specimens, the analysis of viral protein glycoforms, proteomics approaches to understand impacts of infection on host cells, and examinations of point-of-care breath analysis have all been explored. This review organises and illustrates these applications with reference to the many studies that have appeared in the literature since the outbreak. In this respect, those studies in which mass spectrometry has a major role are the focus, and only those which have peer-reviewed have been cited.

Detection and evolution of SARS-CoV-2 coronavirus variants of concern with mass spectrometry.

Mass mapping using high-resolution mass spectrometry has been applied to identify and rapidly distinguish SARS-CoV-2 coronavirus strains across five major variants of concern. Deletions or mutations within the surface spike protein across these variants, which originated in the UK, South Africa, Brazil and India (known as the alpha, beta, gamma and delta variants respectively), lead to associated mass differences in the mass maps. Peptides of unique mass have thus been determined that can be used to identify and distinguish the variants. The same mass map profiles are also utilized to construct phylogenetic trees, without the need for protein (or gene) sequences or their alignment, in order to chart and study viral evolution. The combined strategy offers advantages over conventional PCR-based gene-based approaches exploiting the ease with which protein mass maps can be generated and the speed and sensitivity of mass spectrometric analysis.

Mechanisms of antiviral resistance in influenza neuraminidase revealed by a mass spectrometry based phylonumerics approach.

A mass based phylonumerics approach is shown to be able to investigate the origins of the emergence of antiviral resistance mutations in influenza neuraminidase through a global view of mutational trends. Frequent ancestral and descendant mutations that precede and follow the manifestation of antiviral resistance mutations are identified in N2 neuraminidase. The majority occur in the head region around the active site and drive hydrophilicity changes, primarily through the incorporation or loss of hydroxyl groups. These increase or reduce the accessibility of the site to the bulk solvent. The most frequent ancestral mutations that occur on at least two occasions are I/L307M, G/A414S/T, I312T, I/L307S, P386S and S367N; the latter introducing a glycosylation site. The most frequent descendant mutation, after incorporation of an antiviral resistance mutation, is D/E401G/A. Together with others observed, this restore the protein's hydrophobicity about the active site region that limits entry of a sialic acid or inhibitor molecule and reduces viral fitness. The results of this global in silico phylonumerics study demonstrate that evolutionary mechanisms and functionally linked or compensatory mutations, remote in the sequence or structure, can be identified and interrogated at a molecular level.

Ancestral and Compensatory Mutations that Promote Antiviral Resistance in Influenza N1 Neuraminidase Revealed by a Phylonumerics Approach.

Implementation of a new phylonumerics approach to construct a mass tree representing over 6000 H1N1 human influenza strains has enabled ancestral and compensatory descendant mutations to be identified in N1 neuraminidase that promote antiviral resistance and restore viral fitness. Adjacent to the H275Y resistance mutation site, mutations S299A and S247N, respectively, lead the evolution of oseltamivir-resistant strains and restore viral fitness to those strains thereafter. Importantly the mass tree phylonumerics approach can identify such mutations globally, without any positional bias, so that functionally linked or compensatory mutations remote in the sequence or structure of the protein can be identified and interrogated. This is achieved using mass map datasets commonly employed for protein identification in proteomics applications, thus avoiding the need for either gene or protein sequences that are central to other phylogenetic methods.

Identification of epistatic mutations and insights into the evolution of the influenza virus using a mass-based protein phylogenetic approach.

A mass-based protein phylogenetic approach developed in this laboratory has been applied to study mutation trends and identify consecutive or near-consecutive mutations typically associated with positive epistasis. While epistasis is thought to occur commonly during the evolution of viruses, the extent of epistasis in influenza, and its role in the evolution of immune escape and drug resistant mutants, remains to be systematically investigated. Here putative epistatic mutations within H3 hemagglutinin in type A influenza are identified where leading parent mutations were found to predominate within reported antigenic sites of the protein. Frequent subsequent mutations resided exclusively in different antigenic regions, providing the virus with a possible immune escape mechanism, or at other remote sites that drive beneficial protein structural and functional change. The results also enable a "small steps" evolutionary model to be proposed where the more frequent consecutive, or near-consecutive, non-conservative mutations exhibited less structural, and thus functional, change. This favours the evolutionary survival of the virus over mutations associated with more substantive change that may cause or risk its own extinction.

Indirect study of non-covalent protein complexes by MALDI mass spectrometry: Origins, advantages, and applications of the "intensity-fading" approach.

This review article describes the origins, advantages, and application of an indirect approach with which to study protein and other macromolecular complexes and identify the nature and site of interaction interfaces by means of conventional matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). First reported in 1999, it involves the detection of ion depletion or the absence of ions associated with a binding partner or domain in the MALDI mass spectrum of a mixture of interacting components compared to that for an untreated control. Later referred to as intensity-fading in some applications, the method offers numerous advantages over the direct detection of protein and other macromolecule complexes by MALDI-MS and even electrospray ionization (ESI) MS. The origins of this indirect method, its development for use with gel-separated components, validation using companion biochemical assays, and application to a range of protein-antibody and protein-drug complexes are reviewed together with software specifically developed to aid with data interpretation. The sensitivity of the approach for revealing how subtle differences in the structure of the binding partners can be detected by MALDI-MS is also demonstrated. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 35:559-573, 2016.

Mutational analysis employing a phylogenetic mass tree approach in a study of the evolution of the influenza virus.

A mass based approach has been advanced to enable mutations associated with the evolution of proteins to be both charted and interrogated using phylogenetic trees built solely from the masses of peptides generated upon protein proteolysis. The modified MassTree algorithm identifies and displays all such mutations and calculates the frequency of a particular mutation across a tree. Its significance in terms of its position(s) on the tree is scored, where mutations that occur toward the basis of the tree are weighted more favourably. A comparison with data generated from a conventional sequence based tree demonstrates the reliability of mutational analysis employing this approach. Although illustrated for the study of the evolution of influenza hemagglutinin in this work, the approach has far broader applicability and can be applied to investigate the evolution of any organism. In the case of simple microorganisms this can be achieved even without the separation of component proteins. Given the central role that mass map or fingerprint data plays in protein identification in proteomics, this work demonstrates that such data can be successfully employed in a phylogenetics strategy to better understand and predict future evolutionary trends from the perspective of functional proteins expressed by the organism.

Effect of charge on the conformation of highly basic peptides including the tail regions of histone proteins by ion mobility mass spectrometry.

The first systematic and comprehensive study of the charging behaviour and effect of charge on the conformation of specifically constructed arginine-rich peptides and its significance to the N- and C-terminal basic tail regions of histone proteins was conducted using ion mobility mass spectrometry (IM-MS). Among the basic amino acids, arginine has the greatest impact on the charging behaviour and structures of gas phase ions by virtue of its high proton affinity. A close linear correlation was found between either the maximum charge, or most abundant charge state, that the peptides support and their average collision cross section (CCS) values measured by ion mobility mass spectrometry. The calculated collision cross sections for the lowest energy solution state models predicted by the PEP-FOLD algorithm using a modified MOBCAL trajectory method were found to best correlate with the values measured by IM-MS. In the case of the histone peptides, more compact folded structures, supporting less than the maximum number of charges, were observed. These results are consistent with those previously reported for histone dimers where neutralization of the charge at the basic residues of the tail regions did not affect their CCS values.

Stability of the ßB2B3 crystallin heterodimer to increased oxidation by radical probe and ion mobility mass spectrometry.

Ion mobility mass spectrometry was employed to study the structure of the ßB2B3-crystallin heterodimer following oxidation through its increased exposure to hydroxyl radicals. The results demonstrate that the heterodimer can withstand limited oxidation through the incorporation of up to some 10 oxygen atoms per subunit protein without any appreciable change to its average collision cross section and thus conformation. These results are in accord with the oxidation levels and timescales applicable to radical probe mass spectrometry (RP-MS) based protein footprinting experiments. Following prolonged exposure, the heterodimer is increasingly degraded through cleavage of the backbone of the subunit crystallins rather than denaturation such that heterodimeric structures with altered conformations and ion mobilities were not detected. However, evidence from measurements of oxidation levels within peptide segments, suggest the presence of some aggregated structure involving C-terminal domain segments of ßB3 crystallin across residues 115-126 and 152-166. The results demonstrate, for the first time, the ability of ion mobility in conjunction with RP-MS to investigate the stability of protein complexes to, and the onset of, free radical based oxidative damage that has important implications in cataractogenesis.

Advances in radical probe mass spectrometry for protein footprinting in chemical biology applications.

Radical Probe Mass Spectrometry (RP-MS), first introduced in 1999, utilizes hydroxyl radicals generated directly within aqueous solutions using synchrotron radiolysis, electrical discharge, and photochemical laser sources to probe protein structures and their interactions. It achieves this on millisecond and submillisecond timescales that can be used to capture protein dynamics and folding events. Hydroxyl radicals are ideal probes of solvent accessibility as their size approximates a water molecule. Their high reactivity results in oxidation at a multitude of amino acid side chains providing greater structural information than a chemical cross-linker that reacts with only one or few residues. The oxidation of amino acid side chains occurs at rates in accord with the solvent accessibility of the residue so that the extent of oxidation can be quantified to reveal a three-dimensional map or footprint of the protein's surface. Mass spectrometry is central to this analysis of chemical oxidative labelling. This tutorial review, some 15 years on from the first reports, highlights the development and significant growth of the application of RP-MS including its validation and utility with ion-mobility mass spectrometry (IM-MS), the use of RP-MS data to help model protein complexes, studies of the onset of oxidative damage, and more recent advances that enable high throughput applications through simultaneous protein oxidation and on-plate deposition. The accessibility of the RP-MS technology, by means of a modified electrospray ionization source, enables the approach to be implemented in many laboratories to address a wide range of applications in chemical biology.

Evolution of influenza neuraminidase and the detection of antiviral resistant strains using mass trees.

A new approach employing mass trees is described and implemented which enables the evolution of influenza neuraminidase across all subtypes (N1-N9) in human and animal hosts to be monitored and charted without gene or protein sequencing. These mass trees are shown to be congruent with sequence based trees. Such trees can be built solely from the masses of the proteolytic peptide ions of viral proteins recorded by a mass spectrometer. They are shown to be able to correctly chart the evolutionary history of human pandemic influenza viruses, which originated in animal hosts, and can also resolve antiviral resistant from sensitive strains. Furthermore, experimental mass map data recorded for a circulating strain is correctly positioned onto a mass tree so as to quickly establish its evolutionary history and identify whether it is resistant or sensitive to the antiviral inhibitor oseltamivir. This new computational approach is expected to find wider application for evolutionary studies of organisms more generally.

Incorporation of a proteotyping approach using mass spectrometry for surveillance of influenza virus in cell-cultured strains.

The reemergence of deadly pandemic influenza virus strains has necessitated the development of improved methods for rapid detection and subtyping of influenza viruses that will enable more strains to be characterized at the molecular level. Representative circulating strains of human influenza viruses from primary clinical specimens were grown in cell culture, purified through polyethylene glycol precipitation, proteolytically digested with an endoproteinase, and analyzed and identified by high-resolution mass spectrometry using unique signature peptides that are characteristic of type A H1N1 and H3N2 and type B influenza viruses. This proteotyping approach enabled circulating strains of type A influenza virus to be typed and subtyped, cocirculating seasonal and pandemic H1N1 viruses to be differentiated, and the lineage of type B viruses to be determined through single-ion detection by high-resolution mass spectrometry. Results were obtained using virus titers comparable to those used in reverse transcription (RT)-PCR assays with clinical specimens grown in cell cultures. The methodology represents a more rapid and direct approach than RT-PCR and can be integrated into existing procedures currently used for the surveillance of emerging pandemic and seasonal influenza viruses.