Targeted assay for quantification of proteins from the SARSCoV- 2 coronavirus

Importance of the topic

The COVID-19 pandemic has highlighted the need for complementary analytical strategies to nucleic acid tests. Protein-based detection methods can offer direct measurement of viral particles and support high-throughput workflows in clinical and research laboratories. Mass spectrometry-based assays targeting signature peptides from SARS-CoV-2 proteins promise rapid, sensitive, and quantitative analysis of viral load in patient samples.

Study objectives and overview

This work aimed to develop and optimize a targeted peptide quantification assay for two structural proteins of SARS-CoV-2: the spike (S) glycoprotein and the nucleocapsid (N) protein. Recombinant proteins were digested, key peptides were selected, and MS parameters were fine-tuned. The assay was then evaluated by spiking these proteins into pooled nasopharyngeal swab matrices stored in universal transport medium (UTM) to establish sensitivity metrics.

Methodology and Instrumentation

The workflow comprised:

• Sample preparation: Trypsin/LysC digestion of recombinant S and N proteins, followed by acetone precipitation of spiked UTM swab samples and repeat digestion.
• Chromatography: Fast peptide separation on an ExionLC System using a Phenomenex Luna Omega Polar C18 column (2.1×100 mm, 3 µm) at 0.6 mL/min with a simple gradient (0–90% acetonitrile over 6 min).
• Mass spectrometry: Scheduled MRM on a SCIEX Triple Quad™ 5500+ QTRAP® in positive ionization mode (ISV 5500 V, GS1 60, GS2 50, TEM 600 °C, CUR 30), 40 s detection window, 0.5 s cycle time.
• Data processing: Transition optimization with Skyline and quantitative processing in SCIEX OS-Q software.

Instrumentation used

• SCIEX ExionLC™ System
• Phenomenex Luna Omega Polar C18 column (2.1×100 mm, 3 µm)
• SCIEX Triple Quad™ 5500+ System – QTRAP® Ready
• Skyline software and SCIEX OS-Q

Main results and discussion

Fourteen signature peptides (7 per protein) were finalized based on signal intensity, signal-to-noise ratio, and specificity in UTM. Optimized MRM transitions (two per peptide) yielded sharp chromatographic peaks and robust detection. The lower limits of quantification (LLOQ) ranged from 0.14 to 4.4 fmol/µL in UTM, with the best peptides achieving LLOQs below 0.3 fmol/µL and calibration curves exhibiting r2 ≥ 0.99 for most targets.

Contributions and practical applications

This assay demonstrates a rapid (7 min run), high-throughput approach for direct quantification of SARS-CoV-2 proteins in clinical sample matrices. It can serve as a research tool for viral load studies, support QA/QC in vaccine production, and lay the groundwork for protein-based diagnostic methods as an alternative to PCR.

Future trends and potential applications

Further efforts will focus on validating the assay in authentic patient specimens and diverse transport media. Incorporation of stable isotope-labeled peptide standards will enhance quantitative accuracy. Future developments may integrate immunoaffinity enrichment, automated sample prep, increased multiplexing, and lower detection limits to support routine diagnostic workflows.

Conclusion

A targeted MRM assay for SARS-CoV-2 spike and nucleocapsid proteins was successfully developed on the SCIEX Triple Quad 5500+ QTRAP platform. The method achieves sub-femtomole sensitivity in UTM, rapid analysis times, and robust quantitative performance. Validation in clinical samples and standardization with isotope-labeled standards will be critical for translation to diagnostic applications.

References

1. King J, Kosinski-Collins M, Sundberg E. Coronavirus Structure, Vaccine and Therapy Development.