
==== Front
J Mass Spectrom Adv Clin Lab
J Mass Spectrom Adv Clin Lab
Journal of Mass Spectrometry and Advances in the Clinical Lab
2667-1468
2667-145X
Elsevier

S2667-145X(24)00029-4
10.1016/j.jmsacl.2024.08.003
Research Article
Rapid identification of SARS CoV-2 omicron sub-variant JN.1 (BA.2.86.1.1) with mass spectrometry
Lanyon Henry E.
Downard Kevin M. kevin.downard@scientia.org.au
⁎
Infectious Disease Responses Laboratory, Prince of Wales Clinical Research Sciences, NSW, Sydney, Australia
⁎ Corresponding author. kevin.downard@scientia.org.au
11 8 2024
8 2024
11 8 2024
33 3842
3 4 2024
7 8 2024
10 8 2024
© 2024 THE AUTHORS
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• Rapid Identification of new SARS CoV-2 omicron sub-variant JN.1 virus.

• Mass spectrometry detects L455S mutation peptide biomarker in clinical specimens.

• Differentiation of JN.1 variant from other BA.2.86 strains impacting global health.

Objective

The rapid detection and differentiation of strains of the BA.2.86 lineage including the new sub-variant JN.1 (BA.2.86.1.1) is demonstrated employing selected ion monitoring (SIM) and high resolution mass spectrometry.

Methods

A study of a preliminary set of BA.2.86 lineage positive specimens, identified BA.2.86 and BA.2.86.1.1 peptide markers in 62.5 % and 29.1 % of samples.

Results

Peptide-specific markers in the surface spike protein associated with the L455S mutation are confidently detected with high sensitivity in protein and virus digests.

The virus was thus confidently assigned in over 91 % of positive specimens.

Conclusions

A rise in the global prevalence of the JN.1 (BA.2.86.1.1) immune evasive sub-variant, that emerged in late 2023, requires that new strategies and protocols to detect such strains in human specimens are accelerated and implemented.

Keywords

SARS CoV-2
Coronavirus
Omicron
JN.1
Mass spectrometry
Abbreviations

ACE2 angiotensin-converting enzyme 2

FT-ICR Fourier-transform ion cyclotron resonance

HR-MS High-resolution mass spectrometry

RBD receptor binding domain

SARS-CoV-2 severe acute respiratory syndrome coronavirus

SIM selected ion monitoring

MALDI Matrix-Assisted Laser Desorption Ionization
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pmcIntroduction

Following a period of recombination of Omicron variants of SARS-CoV-2 during 2022 and 2023 [1], [2], a new phylogenetically distinct sub-variant of a previous Omicron strain (BA.2.86) has evolved and taken hold in the past year. As of 20 November 2023, over 3200 nucleotide sequences for BA.2.86 variants were submitted to the Global Initiative on Sharing All Influenza Database (GISAID) from nearly 50 countries, representing around 9 % of the total number of sequences for that period. Despite the growth in prevalence of this variant, the World Health Organization (WHO) declared the public health risk posed by BA.2.86 strains to be low at the global level.

A new BA.2.86.1.1 (or JN.1) sub-variant emerged in late 2023 and was classified as a variant of interest by the WHO in December of that year [3]. Compared with recombinant XBB.1.5, and earlier BA.2 Omicron variants, BA.2.86.1.1 exhibits more than 30 mutations across its spike protein sequence that significantly contribute to its ability to evade a human’s immune system defences [4].

The JN.1 variant has proven to be more challenging to neutralize than its ancestral BA.2.86 subtype [5]. Notably, the JN.1 variant has exhibited strong resistance to immunity provided by the XBB.1.5 vaccine [5], making it one of the most immune-evading variants identified to date. By the end of 2023, the JN.1 virus instigated a significant shift in the landscape of circulating SARS-CoV-2 variants. It has emerged as the dominant strain in Australia and has been linked to a marked increase in hospitalizations. Globally, JN.1 variants are driving the most substantial wave of SARS-CoV-2 observed in many jurisdictions for at least the past year.

The distinctive L455S mutation, unique to the JN.1 variant, has been associated with the virus's enhanced transmissibility and its ability to evade human immune responses [6]. Situated within the receptor binding domain (RBD), this mutation has been shown to enhance the binding activity of the spike protein with the human angiotensin-converting enzyme 2 (ACE2) [7].

This peptide segment, containing the L455S mutation, serves as a useful marker for identifying this variant and distinguishing it from BA.2.86 strains using mass spectrometry. High-resolution mass spectrometry (HR-MS) is particularly advantageous in this context, as it can confidently detect peptides in protein or whole virus digests without the necessity for tandem mass spectrometric sequencing, thereby saving time and enhancing the sensitivity of analysis [8]. This method provides a time and sensitivity advantage, especially when selected ion monitoring (SIM) is employed. Utilizing a proteotyping approach, each of the five major variants of concern (Alpha, Beta, Gamma, Delta, and original Omicron) [9], as well as Omicron sub-variants [10] and recombinant variants, can be identified with high confidence based on mass spectrometry data alone, enabling detection and differentiation [11]. Here, we demonstrate the detection of the distinctive L455S mutation in the surface spike protein using a recombinant protein digest and a preliminary set of clinical specimens.

Materials and methods

Recombinant protein digestion

A C-terminal His(x10)-tagged recombinant form of the spike protein (>95 % purity by gel electrophoresis) derived from a SARS-CoV-2 BA.2.86 strain (Denmark/DCGC-647646/2023) (GISAID ID: EPI_ISL_18097315) isolated on 31/0723, encompassing residues 20–1213, was purchased from Acro Biosystems (Newark, DE, USA). The protein (50 μg) was solubilised in 100 μL of digestion buffer (50 mM ammonium bicarbonate, 10 % acetonitrile, 2 mM dithiothreitol), incubated for 2.5 h at 37 °C, and digested overnight (∼15 h) following the addition of 1 μg of sequencing-grade endoproteinase Asp-N (Merck, Macquarie Park, Sydney, NSW, Australia) (1:50 enzyme:protein) in 1 μL of buffer.

Clinical specimen processing and whole virus digestion

All procedures related to the collection and preparation of specimens were conducted in compliance with the Communicable Diseases Network Australia (CDNA) national guidelines for Coronavirus Disease 2019 and NSW Health restrictions and protocols. Human clinical specimens were sourced from the Prince of Wales or Westmead Hospitals, Sydney, as nasopharyngeal swabs. A series of twenty-four (24) clinical specimens identified by RT-PCR as containing SARS-CoV-2 Omicron strains of the BA.2.86 lineage (BA.2.86 or BA.2.86.1.1) and an additional six SARS-CoV-2 negative control specimens were utilized. These specimens were collected with consent from patients infected with Omicron SARS-CoV-2 and, along with the negative controls, were placed in a saline solution (1 mL) and stored at −70 °C. To enhance precision and reduce biases, consistent pre-analytical sample handling and pre-purification techniques were applied to all samples. RNA was extracted from half (0.5 mL) of the solution and quantified by RT-qPCR following a reported protocol [12] with virus titers found to be between 105–106 copies/mL in the positive specimens. The presence of the BA.2.86-specific S-gene insertion (for ins16-MPLF) in these samples was identified using insertion-specific primers in a documented dsDNA dye-based PCR hybridization procedure [13].

Virus was precipitated from the remaining solution using a 95 % ethanol solution at −20 °C, captured on a 300 kDa molecular weight cut-off (MWCO) filter (Pall Corporation, Cheltenham, Victoria, Australia), washed with purified water, and the retentate recovered. The virus was resuspended in 100 μL of digestion buffer (50 mM ammonium bicarbonate, 10 % acetonitrile, 2 mM dithiothreitol, and 5 mM octyl β-D-glucopyranoside) at pH 7.5. The solution was sonicated for 15 min, incubated for 2 h at 37 °C, and then digested overnight with the addition of 15 ng/μL sequencing-grade endoproteinase Asp-N (Merck, Macquarie Park, Sydney, NSW, Australia).

High-resolution MALDI-FT-ICR mass spectrometry

The viral peptide solutions were desalted using ZipTip C(18) pipette tips (Merck, Macquarie Park, Sydney, NSW, Australia). An aliquot (1 μL) was diluted with a matrix solution (10 μl) containing 5 mg/mL α-cyano-4-hydroxycinnamic acid in 50 % acetonitrile with 0.1 % trifluoroacetic acid. Solution volumes of 1 μL of were spotted onto a Matrix-Assisted Laser Desorption Ionization (MALDI) sample plate and analyzed using a Bruker SolariX 7 T XR Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with a MALDI source (Bruker Daltonics, Preston Victoria, Australia). Spectra were acquired across a mass-to-charge ratio (m/z) range of 750–3250 using broadband excitation or a narrow band range of 20 mDa (+/-0.01 m/z) for selected ion monitoring (SIM) [16] centred on the theoretical (monoisotopic) values for each of the respective protonated peptide ions. The instrument was externally calibrated with a standard peptide mixture. Spike protein peptides were matched to predicted proteolytic products generated in silico using the Expasy PeptideMass tool (https://web.expasy.org/peptide_mass/) considering peptides with a mass greater than 500, no modifications, and allowing for a maximum of one missed cleavage site.

BLAST (protein) search of the SARS-CoV-2 viral and human proteome with mutation-containing S1 peptides

The S1 peptides comprising residues 450–464/6 that include the L455S mutation site for both the BA.2.86 and BA.2.86.1.1 (JN.1) variants (with sequence DYWYRL/SFRKSKLKPF(+ER)) were separately examined for their uniqueness among known proteins of the SARS-CoV-2 virus (taxid:2697049), all coronaviruses (taxid:11118), and the human (Homo sapiens) (taxid:9606) proteome. This was performed using a Basic Local Alignment Search Tool Protein (BLASTp) search (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins; conducted on 26/03/24) of the non-redundant protein sequence database for this host organism. All four peptides were determined to be unique to the human spike protein of SARS-CoV-2, showing 100% coverage.

Results and discussion

Spike protein sequences for BA.2.86 and BA.2.86.1.1 (JN.1) and their digest implications

The characteristic spike protein mutations of the JN.1 (BA.2.86.1.1) variant are outlined in Table 1, alongside those identified in the earlier BA.2 subtype and the circulating XBB.1.5 variant, a recombinant form of the BA.2.10 and BA.2.75 strains. JN.1 is closely related to the BA.2.86 variant. It differs only by the presence of an additional single mutation. The L455S mutation, located within the receptor binding domain (RBD) region atop the protein, is specific to the JN.1 (BA.2.86.1.1) variant and serves as a potential marker to distinguish such strains.

Fig. 1 illustrates that this mutation is situated in an area rich in lysine and arginine residues. Consequently, a series of short peptides (shown in red) spanning residues 455–457, 458, and 459–460 are generated upon full cleavage with trypsin. In contrast, cleavage with endoproteinase Glu-C produces a larger peptide encompassing residues 406–465 (shown in blue). The small peptides are less detectable by mass spectrometry since they can be obscured by matrix, buffers, and other contaminants in the low mass range of a spectrum while the large peptides exhibit reduced detection efficiency in the high mass range (>5000 Da.) on most instruments.Fig. 1 Partial sequence of the spike protein of a BA.2.86 strain spanning residues 405–465 of the receptor binding domain (RBD) showing the position of the L455S mutation (of the BA.2.86.1.1/JN.1 sub-variant) and trypsin, Glu-C and Asp-N generated peptides that contain it or which are in its vicinity.

To circumvent these issues, an endoproteinase Asp-N digest was employed. This protease cleaves peptide bonds on the N-terminal side of aspartic and glutamic acid residues, and generates a segment comprising residues 450–464 (Fig. 1, green). This 15-residue peptide (with sequence DYWYRLFRKSKLKPF) has a theoretical monoisotopic mass of 2047.1276 and 2021.0755 for the BA.2.86 and BA.2.86.1.1 (JN.1) strains, respectively. Being a highly basic peptide containing two arginine and three lysine residues, its ionization by MALDI is aided in the positive ion mode and its mass falls within the mid-spectral range typical for a peptide digest to facilitatie detection.

MALDI mass spectrum of an Asp-N digest of the spike protein of a BA.2.86 strain

The high-resolution MALDI mass spectrum of an Asp-N digest of the recombinant spike protein for a BA.2.86 strain collected in Denmark in mid-2023 is shown in Fig. 2. Spanning a m/z range of 750–3250, the spectrum encompasses peptides associated with both the S1 (Appendix A, [14], [15]) and S2 subunits (underlined). Among them is an intense ion at m/z 2047.1295 (+0.93 ppm) that is linked to S1 residues 450–464, covering the 455 mutation site. A second peptide detected at m/z 2332.2731 (+0.77 ppm) corresponds to a miscleaved peptide comprising residues 450–466, with additional ER residues at the C-terminus. A mass resolution m/△m (FWHM) of 22,401 was obtained for the ions at m/z 2332.2731. Either peptide can be used to confirm the presence of a BA.2.86 strain. The simultaneous presence of both peptides, as observed in Fig. 2, provides greater support.Fig. 2 High resolution MALDI mass map spectra of the endoproteinase Asp-N digest of recombinant spike protein subunits of a BA.2.86 strain. Numbering is in accordance to the recombinant sequence which comprises residues 20–1213 coupled to a polyhistidine tag at the C-terminus. Residues denoted with an asterisk (*) denote miscleaved products while those underlined are located in the S2 subunit. Mass resolution (FWHM) measured at m/z 2332 is 22401.

The masses of these peptides, along with the equivalent peptides where leucine is substituted by serine at position 455, at m/z 2021.0755 and 2306.2192 (theoretical), were utilised to detect and differentiate BA.2.86 and BA.2.86.1.1 (JN.1) strains within a preliminary set of clinical specimens.

Detection of sub-variant specific markers in clinical specimens

The collective four peptides were used to identify and differentiate BA.2.86 and BA.2.86.1.1 (JN.1) strains in a set of 24 clinical specimens. These were identified by RT-PCR as containing SARS-CoV-2 Omicron strains of the BA.2.86 lineage, and were used alongside six negative control specimens.

To optimize detection, SIM was utilized to reduce acquisition time and thereby enhance sensitivity by focussing solely on the four peptide markers of interest. The relative abundances of each peptide detected in the positive and negative control samples are plotted in Fig. 3. The intensities recorded in the positive specimens for each peptide ion, surpassing the highest value observed in the negative control, were plotted alongside the control values. The line headers represent the maximum and minimum intensities, while the box edges indicate the first and third quartile intensities. The central line for each peptide signifies the median value.Fig. 3 Box plots of the measured intensities of the four peptide biomarkers for BA.2.86 at m/z 2047.1276 and 2332.2713, and BA.2.86.1.1 (JN.1) at m/z 2021.0755 and 2306.2192 in positive (24) and negative control (6) specimens. The lines indicated the maximum and minimum intensities, the box edges indicate the first and third quartile of intensities, while central line indicates the median value. The number of specimens detected, or not (for negative controls), for each ions is shown in brackets.

Among the 24 positive samples for BA.2.86, ions at both m/z 2047.1276 (±0.01) and 2332.2713 (±0.01), associated with S1 residues 450–464/466 spanning the 455 mutation site, were detected with intensities exceeding the control in 15 of the specimens. In six additional samples, ions at m/z 2021.0755 and 2306.2192 corresponding to BA.2.86.1.1 (JN.1) strains were identified. In one additional specimen, only the former ion was detected above control intensities. Consequently, 62.5 % of positive specimens were classified as BA.2.86 strains, and 29.1 % were identified as BA.2.86.1.1 strains, with 25 % more confidently assigned based upon the detection of both markers.

For two RT-PCR positive specimens, no BA.2.86 lineage peptide markers were detected. This may be attributed to virus loads below detectable levels with mass spectrometry, or the presence of other peptides unique to these strains, or contaminants that suppress the BA.2.86 lineage ions. This corresponds to a 8.3 % false negative rate. No BA.2.86 lineage-specific marker peptides were detected in the negative controls, resulting in no false positive measurements.

Conclusions

The emergence of Omicron sub-variants within the BA.2.86 lineage poses a significant human health risk as they may impact the efficacy of current COVID-19 vaccines and antiviral therapies. The prompt and sensitive detection of these sub-variants is crucial for curtailing their transmission and advancing more effective epidemiological responses and therapies. This study demonstrates that common SARS-CoV-2 Omicron sub-variants within the BA.2.86 lineage can be accurately identified and differentiated using high-resolution mass spectrometry employing a SIM approach. By significantly reducing scan times by 125,000 (for the experimental ranges employed in this study) and thereby enhancing sensitivity, this method offers a valuable advancement in detection capabilities.

The identification of distinct peptide biomarkers associated with the L455S mutation enables the discrimination between BA.2.86 and BA.2.86.1.1 strains without the need for gene or protein sequencing. The utilization of high-resolution MALDI-MS facilitates the more confident discrimination of specimens compared with benchtop MALDI-TOF instruments that exhibit lower mass resolutions and accuracies [17], [18].

The novel mass spectrometry-based strategy [19], [20] described provides a promising alternative to RT-PCR-based analyses [21]. This methodology streamlines the analysis process, with virus digestion from clinical specimens conducted within a comparable timeframe to the numerous steps required by the workflow to isolate, purify and amplify viral RNA where total analysis times take at least 24 − 48 h. Furthermore, the rapid evolution of the virus requires that the sequences of primers and probes be continuously monitored, otherwise any RT-PCR approach can fail. High false-negative rates have been reported in some SARS-CoV-2 RT-PCR studies [22].

In contrast, the MS clinical workflow [19], [20] described here requires no separation of viral components and allows for swift pretreatment and proteolytic digestion of samples, facilitating rapid mass spectra acquisition from multiple digested virus samples. This streamlined MALDI approach, coupled with automated or semi-automated spectral acquisition, enables the analysis of hundreds of samples in a short time span, with each spectrum acquired in seconds and is inherently faster than LC-MS methods.

While PCR methods offer sensitivity advantages through the amplification of viral RNA, MS analysis's sensitivity is contingent on the actual viral protein levels in the sample. This provides PCR-based methods with a sensitivity advantage down to ∼100–1000 copies of virus, while MS approaches typically require 5000–10,000 copies, as demonstrated by our earlier studies on both SARS-CoV-2 [19], [20] and influenza [23]. Despite the initial cost of a high-resolution mass spectrometer, which is required to confidently assign viral peptides from mass map data without any MS/MS sequencing, its usage is a one-time investment and aligns with the expenses associated with the purchase of multiple PCR sequencers and associated equipment.

Finally, such protein-based approaches are more transferable to studies of viral proteins to assess infectivity and immune evasion mechanisms, as well as in developing monoclonal antibody therapies and antiviral drug therapies [24]. Thus protein mass spectrometry approaches represent a valuable technology for virological studies and therapeutic development.

Ethics statement

All procedures involved in collection and preparation of the specimens were carried out in accordance with the Communicable Diseases Network Australia (CDNA) national guidelines for Coronavirus Disease 2019 and NSW Health restrictions and protocols. Anonymised human clinical specimens were sourced with consent from Omicron SARS-CoV-2 infected patients.

Funding information

Support from the Clinical Research Fund (CRF24) to author Downard, and donours to this study, is acknowledged.

CRediT authorship contribution statement

Henry E. Lanyon: Methodology, Investigation, Formal analysis, Data curation. Kevin M. Downard: Conceptualization, Methodology, Investigation, Writing – original draft, Resources, Supervision, Writing – review & editing, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following are the Supplementary data to this article:Supplementary Data 1

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.jmsacl.2024.08.003.
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