LC-MS for detection of SARS-CoV-2 viral and host proteins

Significance of the Topic

This study addresses the need for complementary methods to RT-PCR and antigen tests in COVID-19 diagnosis and prognosis.
Mass spectrometry offers high specificity for detecting viral proteins and quantifying host response markers, supporting clinical decisions on disease severity and progression.

Objectives and Study Overview

The primary aim was to develop and validate a robust LC-MS workflow for targeted detection of SARS-CoV-2 peptides and host protein surrogates in nasopharyngeal swab and plasma samples.
The study compared non-severe and severe COVID-19 cohorts to identify prognostic biomarkers and assess correlation with viral load.

Methodology and Instrumentation

Sample Preparation and Discovery Proteomics

• Plasma: depletion of abundant proteins, reduction/alkylation, trypsin digestion.
• Swabs: heat inactivation, organic solvent precipitation, digestion.
• Desalting via C18 cleanup.

Chromatography and Mass Spectrometry

• Discovery: EASY-nLC 1200 coupled to Orbitrap Fusion Lumos (HRAM) in data-dependent mode.
• Targeted SRM: Vanquish Flex UHPLC and TSQ Altis triple quadrupole using optimized gradients on Hypersil GOLD C18 columns.

Instrument List

• Thermo Scientific Orbitrap Fusion Lumos Tribrid MS
• Thermo Scientific TSQ Altis Triple Quadrupole MS
• Thermo Scientific Vanquish Flex Quaternary UHPLC
• Thermo Scientific EASY-nLC 1200 nanoLC system

Key Findings and Discussion

Plasma Proteome

• Label-free quantification detected ~1,200 proteins; 38 showed significant changes between severe and non-severe groups.
• Up-regulated in severe cases: fibrinogen gamma chain (FGG), SERPINA4, Serum amyloid P-component (APCS), S100A8.
• Down-regulated: complement factors D and C8A, CD14.
• Targeted SRM validated overexpression of host markers (e.g., angiotensinogen, apolipoprotein B) with synthetic heavy peptide controls ensuring run consistency.

Swab Proteome and Viral Peptides

• Shotgun analysis on pooled solvents maximized viral peptide coverage.
• MRM assays monitored 11 peptides from nucleoprotein, spike glycoprotein, and replicase polyprotein 1ab.
• Distinct elution profiles allowed sensitive discrimination of COVID-19 positive and negative samples.
• Host inflammation markers (LDH, IL-6, CRP) also detected in swabs, correlating with disease status.

Benefits and Practical Applications

• Provides orthogonal confirmation to RT-PCR, reducing false results.
• Quantitative readout of viral load and host response enables severity assessment.
• Multiplexed assay can be implemented on existing high-flow LC-MS platforms for clinical research.

Future Trends and Potential Applications

• Expansion to larger, diverse cohorts with blinded validation.
• Development of simplified, high-throughput SRM panels for point-of-care research labs.
• Integration into multi-omics pipelines to monitor emerging variants and host pathways.
• Application to other respiratory pathogens using similar proteomic workflows.

Conclusion

Combining discovery and targeted LC-MS approaches enabled sensitive detection of SARS-CoV-2 peptides and host prognostic markers in swab and plasma samples.
The workflow demonstrates potential for clinical research applications in diagnosis, severity monitoring, and biomarker discovery beyond conventional methods.

References

1. Tang Y, Schmitz JE, Persing DH, Stratton CW. Laboratory Diagnosis of COVID-19: Current Issues and Challenges. J Clin Microbiol. 2020;58(6):e00512-20.
2. Preprint: COVID-19 plasma deep proteome reveals distinct signatures in severe patients. Research Square. 2021.
3. Ponnusamy D, et al. Proteomic investigation reveals dominant alterations of neutrophil degranulation and mRNA translation pathways in COVID-19 patients. PLoS ONE. 2020;15(11):e0240012.