Analysis of SARS-CoV-2 using LC-MS Peptide Enrichment for Clinical Research

Significance of the topic

Detection of SARS-CoV-2 proteins at low levels is crucial for understanding viral replication, monitoring infection progression and supporting longitudinal clinical research. Unlike PCR, which infers viral presence through nucleic acid amplification, direct protein analysis by LC-MS/MS coupled with peptide enrichment offers higher specificity, quantification accuracy and the ability to monitor multiple targets simultaneously.

Study objectives and overview

This study aimed to develop and validate an automated workflow for quantitative detection of SARS-CoV-2 nucleocapsid peptides using SISCAPA immunoaffinity enrichment combined with LC-MS/MS. The approach was tested in viral transport medium with synthetic peptide calibrators and spiked nucleocapsid protein to assess sensitivity, precision, linearity and variant discrimination.

Methodology and instrumentation

The workflow comprises four main steps: protein denaturation and tryptic digestion, peptide enrichment, LC-MS/MS analysis and data processing. Key elements include:

• Automated sample preparation on an Andrew Alliance Andrew+ pipetting robot with OneLab software
• SISCAPA magnetic beads coated with anti-peptide antibodies for enrichment of three nucleocapsid peptides (ADE, AYN, NPA)
• Quantitative internal standards using stable isotope labeled peptides
• Peptide separation on Waters ACQUITY Premier Peptide BEH 300Å C18 column (1.7 µm, 2.1 x 30 mm)
• Detection by Waters ACQUITY UPLC I-Class system coupled to a Xevo TQ-XS triple quadrupole mass spectrometer operated in MRM mode
• Overall analysis time per sample of approximately 2.5 minutes

Main results and discussion

Analytical sensitivity was demonstrated with a lower limit of quantification of 3 amol/µL for each peptide, achieving precision within 20%, bias within ±20% and signal-to-noise ratios above 10:1. Calibration curves spanning 3 to 50000 amol/µL showed excellent linearity (r2 >0.99). Intra- and inter-day precision for synthetic peptides was ≤12.4% CV and for nucleocapsid protein-derived peptides ≤18.8% CV. No significant carryover was observed after high-level injections and extracted samples remained stable on the autosampler for at least 48 hours. Matrix effects in PBS and two viral transport media ranged from 94–108%. The method was further extended to detect a Delta variant mutation (D377Y) by adding an additional MRM transition, demonstrating the capacity to distinguish peptide variants chromatographically.

Benefits and practical applications

The validated workflow offers:

• Robust quantification of viral proteins in clinical research samples
• High throughput capability with automated sample preparation and rapid LC-MS/MS runs
• Multiplexed detection of multiple peptides and potential biomarkers in a single analysis
• Flexibility to adapt to emerging viral mutations and variants
• Standardization potential across laboratories through direct protein measurement

Future trends and opportunities

Advances in immunoaffinity enrichment, automation and mass spectrometry will further lower detection limits and increase throughput. Integration with biomarker panels for disease severity could enable comprehensive viral-host interaction studies. Real-time data analysis and cloud-based workflows may accelerate multi-site harmonization. Expanding the method to include additional viral proteins or post-translational modifications will enhance mechanistic insights into SARS-CoV-2 pathogenesis.

Conclusion

The combination of SISCAPA enrichment with high-sensitivity LC-MS/MS provides a powerful platform for direct quantitative detection of SARS-CoV-2 nucleocapsid peptides. The method achieves a 3 amol/µL limit of quantification, strong precision and linearity, minimal matrix effects and the ability to monitor variant-specific peptide changes. This workflow supports rigorous clinical research and cross-laboratory standardization of viral protein analysis.