A quick and robust mass spectrometry-based method for the detection of SARS-CoV-2

Significance of the Topic

The COVID-19 pandemic has highlighted the need for rapid, reliable, and orthogonal diagnostic methods complementing RT-PCR. Mass spectrometry–based peptide quantitation offers direct detection of viral proteins, potential viral load assessment, and adaptability to emerging variants.

Aims and Study Overview

This study aimed to develop and validate a four-minute, bottom-up LC-MS/MS assay on a TSQ Altis MD system to detect and absolutely quantify six signature peptides from the SARS-CoV-2 spike and nucleocapsid proteins in nasopharyngeal swab and saliva matrices.

Methodology

Samples of recombinant spike (P0DTC2) and nucleocapsid (P0DTC9) proteins were spiked into pooled nasal fluids or saliva and stored in viral transport medium. Proteins were precipitated with cold acetone, digested with SMART Digest trypsin, and diluted for LC-MS/MS analysis. Selected reaction monitoring targeted six proteotypic peptides, using stable isotope–labeled internal standards for retention time confirmation and quantification. Calibration ranges spanned 0.01–100 fmol on column.

Used Instrumentation

• Thermo Scientific TSQ Altis MD triple-quadrupole mass spectrometer
• Thermo Scientific Vanquish MD UHPLC system
• Hypersil GOLD C18 column (2.1 × 50 mm, 1.9 μm)
• Thermo Scientific SMART Digest Trypsin Kit
• TraceFinder LDT software for data processing

Main Results and Discussion

The assay achieved limits of detection of 0.25–5 fmol and quantitation limits of 0.5–10 fmol on column across both matrices, with R² values >0.99 and CV/RSD <15%. Peptides from both nucleocapsid and spike proteins exhibited consistent retention times (<±0.01 min), robust linearity, and clear differentiation by mass transitions, enabling reliable identification and quantitation.

Benefits and Practical Applications

• High throughput: 4-minute run time per sample
• Absolute quantitation supports viral load estimation
• Dual-protein targeting reduces false positives
• Automatable data processing for rapid reporting

Future Trends and Applications

The workflow can expand into multiplexed panels for respiratory pathogens (e.g., influenza, RSV) or track variant-specific peptides. Further validation against clinical PCR results and exploration of simplified sample prep could enhance scalability in diagnostic laboratories.

Conclusion

This targeted LC-MS/MS method provides a fast, accurate, and reproducible approach for SARS-CoV-2 protein detection in clinical matrices. Its sensitivity, specificity, and adaptability position it as a valuable complement to existing molecular diagnostics.

Reference

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