Developing a Quick and Robust Mass Spectrometry-based Method for the Detection of SARS-CoV-2

Importance of the Topic

Accurate and rapid detection of SARS-CoV-2 remains critical for controlling outbreaks and supporting public health decisions. Mass spectrometry offers an orthogonal approach to PCR-based diagnostics, addressing reagent shortages and providing precise peptide quantitation as a complementary tool.

Study Objectives and Overview

This work aims to develop a quick and robust bottom-up proteolytic workflow coupled with targeted mass spectrometry to detect SARS-CoV-2 peptides in clinical specimens. Six peptides derived from recombinant viral proteins were selected for optimization and quantitation.

Methodology

Clinical matrices (pooled nasopharyngeal fluids and saliva) were spiked with recombinant SARS-CoV-2 proteins and processed using precipitation, centrifugation, and enzymatic digestion with SMART Digest Trypsin. Peptide separation was achieved via a rapid 4-minute LC gradient on a Hypersil GOLD C18 column. A single reaction monitoring (SRM) method targeted specific precursor-to-product ion transitions for each peptide. Data acquisition and quantitation employed TraceFinder LDT software using stable isotope-labeled standards.

Instrumentation

• Thermo Scientific Vanquish MD HPLC system
• Thermo Scientific TSQ Altis MD mass spectrometer
• Hypersil GOLD C18 column (1.9 µm, 2.1 x 50 mm)
• Thermo Scientific SMART Digest Trypsin kit

Main Results and Discussion

The optimized SRM assay achieved limits of detection between 0.25 and 5.0 fmol on column and limits of quantitation from 0.5 to 10.0 fmol. Five nucleocapsid peptides and one spike peptide demonstrated consistent retention times with minimal variance (<0.01 min) relative to isotope-labeled standards. Calibration curves exhibited linearity across the targeted concentration range, confirming method robustness for absolute quantitation.

Benefits and Practical Applications

• Sub-femtomole sensitivity enables detection in low-abundance samples.
• Rapid 4-minute LC/MS run time supports high-throughput screening.
• Orthogonal confirmation to PCR enhances diagnostic confidence.
• Quantitative output aids in viral load assessment and epidemiological studies.

Future Trends and Opportunities

Expanding the peptide panel may improve coverage across viral variants. Integration of automation and multiplexing could accelerate sample processing. Advances in portable and benchtop mass spectrometry could facilitate point-of-care applications. Combining MS-based assays with microfluidics or immuno-enrichment may further enhance sensitivity.

Conclusion

A robust, fast, and reliable LC/MS-based assay has been established for absolute quantitation of SARS-CoV-2 peptides. This workflow provides a valuable complement to molecular methods, offering high specificity, reproducibility, and throughput suitable for clinical and research laboratories.

References

1. Coronavirus in the US: Latest Map and Case Count. The New York Times, 2020.
2. He J., Guo Y., Mao R., Zhang J., Xu X. Diagnostic Performance Between CT and Initial Real-Time RT-PCR. Respiratory Medicine, 168:105980, 2020.