Including peptide enrichment in a mass spectrometry-based workflow for the absolute quantitation of SARS-CoV-2

Significance of the Topic

This study addresses the urgent need for reliable measurement of SARS-CoV-2 proteins in clinical specimens. Absolute quantitation of viral biomarkers in nasal fluids supports diagnostic assay development, epidemiological monitoring and evaluation of viral load, offering an orthogonal approach to nucleic acid tests.

Objectives and Study Overview

The primary goal was to demonstrate that integrating peptide enrichment into a liquid chromatography–mass spectrometry (LC-MS) workflow enhances sensitivity and throughput for measuring SARS-CoV-2 nucleocapsid protein. Recombinant protein spiked into pooled nasal fluids was used to validate method performance.

Methodology and Instrumentation

Sample preparation included protein precipitation, enzymatic digestion with a SMART Digest™ trypsin kit, and peptide enrichment via SISCAPA using anti-peptide antibodies. Stable isotope-labeled standards (SIS) for each target peptide enabled absolute quantitation and compensation for matrix effects. Separation was achieved on a Hypersil GOLD™ C18 column (2.1×50 mm, 1.9 µm) with a 2-minute gradient. Detection employed a Thermo Scientific™ TSQ Altis™ mass spectrometer operating in selected reaction monitoring (SRM) mode. Data analysis used TraceFinder™ LDT software.

Main Results and Discussion

• Limits of detection ranged from 0.25 to 1.0 fmol on-column; limits of quantitation from 0.25 to 2.5 fmol.
• Calibration curves for five peptides showed linearity with R2 > 0.99 across 0.25–100 fmol.
• Precision was within 15% relative standard deviation across the analytical measurement range.
• Chromatographic peaks were well resolved, with retention time variability below ±0.01 minutes.
• Peptide enrichment reduced background interference and enabled 2-minute LC runs, with each peptide eluting within a 30-second window.

Benefits and Practical Applications

The addition of SISCAPA enrichment significantly improves assay sensitivity, reduces total analysis time and lowers the required sample volume. This approach can be deployed in clinical and research laboratories for high-throughput quantitation of viral proteins, supporting COVID-19 diagnostics, vaccine evaluation and variant surveillance.

Future Trends and Opportunities

Further developments may include multiplexed panels targeting multiple viral proteins or host response markers, integration with automated sample handling for large-scale screening, and adaptation to point-of-care platforms. Advances in high-resolution MS and improved antibody reagents will drive even greater sensitivity and specificity.

Conclusion

Incorporating peptide enrichment into an LC-MS workflow enables robust, high-throughput absolute quantitation of SARS-CoV-2 proteins in nasal fluids, with low femtomole sensitivity and rapid analysis times. This method holds promise for enhancing clinical diagnostics and virological research.

References

1. Coronavirus in the US: Latest Map and Case Count: www.nytimes.com/interactive/2020/us/coronavirus-us-cases.html
2. He J. et al. Diagnostic Performance Between CT and Initial Real-Time RT-PCR for Clinically Suspected 2019 Coronavirus Disease (COVID-19) Patients Outside Wuhan, China. Respir Med. 2020;168:105980.