Comprehending COVID-19: Application of UniSpray and Electrospray Ionization for the Detection of Proteolytic Digested SARS-CoV-2 Proteins

Importance of the Topic

The quantification of SARS-CoV-2 structural proteins by targeted LC-MS offers a direct measure of viral load in clinical samples, complementing nucleic acid tests and providing orthogonal data on infection status. Reliable detection of Spike and Nucleoprotein peptides in nasopharyngeal swabs can support both research and diagnostic workflows, especially when PCR sensitivity or specificity is challenged.

Study Objectives and Overview

This work compares two ionization interfaces—UniSpray and Electrospray (ESI)—on a Xevo TQ-XS tandem quadrupole mass spectrometer for MRM-based detection of tryptic peptides from SARS-CoV-2 Spike and Nucleoprotein in a Universal Transport Medium (UTM) matrix. The goal is to assess signal-to-noise improvements, dynamic range extension, and selectivity benefits of each source.

Methodology

Recombinant SPIKE and NCAP proteins were digested with trypsin-LysC and freeze-dried as standards. Peptides were reconstituted and spiked into UTM to generate a dilution series. Chromatographic separation employed an ACQUITY UPLC I-Class PLUS system with a peptide-optimized C18 column (2.1×50 mm, 1.7 µm) at 40 °C, 0.6 mL/min flow, and an 8-minute gradient of 5–85% acetonitrile (0.1% FA). MRM acquisition on Xevo TQ-XS used two transitions per peptide with optimized collision energies and cone voltages. Data processing utilized MassLynx and TargetLynx.

Used Instrumentation

• ACQUITY UPLC I-Class PLUS System
• ACQUITY PREMIER Peptide BEH C18 Column (2.1×50 mm, 1.7 µm, 300 Å)
• Xevo TQ-XS Tandem Quadrupole MS
• UniSpray and Electrospray ionization sources (positive mode)
• MassLynx and TargetLynx software

Main Results and Discussion

UniSpray demonstrated approximately a 2.5-fold increase in signal-to-noise for the Nucleoprotein peptide ADETQALPQR compared to ESI, enabling reliable quantitation at one additional concentration level without loss of linearity. Across the panel of SPIKE and NCAP peptides, UniSpray yielded on average a four-fold higher peak area. However, a subset of peptides exhibited better response with ESI, underscoring the value of combining both ionization techniques. This complementarity allows selection of the optimal source per peptide to maximize sensitivity and confidence in complex matrices.

Benefits and Practical Applications

• Enhanced sensitivity for low-abundance viral peptides in clinical matrices
• Extended dynamic range enabling quantitation over broader concentration spans
• Flexibility to choose the best ionization method for individual peptides
• Potential to integrate into diagnostic workflows as a confirmatory or complementary assay

Future Trends and Opportunities

Emerging trends include combining both UniSpray and ESI in multiplexed MRM panels for comprehensive viral proteome coverage, adapting workflows to new SARS-CoV-2 variants, automating sample preparation for high throughput, and extending the approach to other pathogens or biomarker targets. Coupling with high-resolution mass spectrometry and data-independent acquisition could further deepen analytical insights.

Conclusion

UniSpray and Electrospray are complementary ionization techniques that together enhance the sensitivity, dynamic range, and selectivity of targeted LC-MS assays for SARS-CoV-2 peptides. Their combined use on the Xevo TQ-XS platform supports robust quantitative measurement of viral proteins in nasopharyngeal swab samples preserved in UTM.

References

• 1. WHO Laboratory Testing for 2019 Novel Coronavirus (2019-nCoV) in Suspected Human Cases. Interim Guidance. Jan 17, 2020.
• 2. Subbaraman N. Coronavirus Tests: Researchers Chase New Diagnostics to Fight the Pandemic. Nature. 2020 Mar 23.
• 3. Dhaenens M. A Universally Adoptable Corona Multiple Reaction Monitoring Assay. Cov-MS Consortium video presentation.
• 4. Bongaerts J et al. A Comparative Study of UniSpray and Electrospray Sources for the Ionization of Neuropeptides in LC-MS/MS. J Chromatogr A. 2020;1628:461462.