Advancing Research with the SARS-CoV-2 LC-MS Kit (RUO)

Significance of the Topic

Quantitative measurement of SARS-CoV-2 proteins is critical to advance clinical research on viral load dynamics, prolonged viral shedding, and prognostic biomarker discovery. Traditional PCR methods amplify genetic material but do not directly measure viral proteins. A reliable LC-MS approach offers direct, multiplexed detection of signature peptides from the viral nucleocapsid, improving specificity and enabling harmonized quantitative workflows across laboratories.

Objectives and Study Overview

This study aims to demonstrate a complete research-use-only LC-MS kit for direct detection and quantification of three SARS-CoV-2 nucleocapsid peptides. Key goals include:

• Automating sample preparation to reduce hands-on time and operator variability.
• Achieving high analytical sensitivity in a complex matrix (viral transport medium).
• Validating linearity, precision, and stability over a broad concentration range.

Methodology

The workflow comprises four main steps:

1. Denaturation and enzymatic digestion of viral proteins to generate target tryptic peptides (AYNVTQAFGR, NPANNAAIVLQLPQGTTLPK, ADETQALPQR).
2. SISCAPA enrichment using anti-peptide antibody-conjugated magnetic beads to concentrate analytes and remove matrix interferences.
3. Automated liquid handling with an Andrew+ Pipetting Robot under OneLab control, standardizing pipetting, incubation, washing, and elution in a 96-well format.
4. Rapid UPLC separation (1.8-min runtime) on a peptide-optimized C18 column, followed by sensitive MRM detection on a triple quadrupole mass spectrometer.

Used Instrumentation

• Andrew+ Pipetting Robot with OneLab software for automated sample prep.
• Waters ACQUITY UPLC I-Class FTN system equipped with Premier Peptide BEH C18, 2.1 × 30 mm, 1.7 µm column.
• Xevo TQ-XS Triple Quadrupole Mass Spectrometer operating in positive ESI mode with MRM acquisition.
• MassLynx v4.2 software with TargetLynx XS for data processing and quantification.

Main Results and Discussion

The method exhibited excellent linearity (r2 > 0.99) across 3–50 000 amol/µL in viral transport medium with 1/x2 weighting. The lower limit of quantification (LLoQ) was established at 3 amol/µL for all three peptides with precision ≤ 17.4% CV and bias within ± 20%. Inter- and intra-day precision remained below 17.1% CV over five days. QC samples were stable on the autosampler at 10 °C for at least 48 hours, confirming robust analytical performance.

Benefits and Practical Applications

• Direct protein quantification without target amplification reduces false positives and assay complexity.
• High sensitivity and selectivity via SISCAPA enrichment enable detection at low amol/µL levels in complex matrices.
• Rapid 2.5-minute cycle time supports high sample throughput for longitudinal research studies.
• Automated sample prep minimizes human error and improves reproducibility across laboratories.
• Multiplex capability allows simultaneous analysis of viral peptides and host biomarkers in one run.

Future Trends and Potential Applications

Advancements may include expanded multiplex panels for variant detection and host response biomarkers, integration into high-throughput clinical research pipelines, and further automation of end-to-end workflows. Emerging mass spectrometry platforms with enhanced speed and sensitivity could extend applicability to other viral pathogens and large-scale surveillance studies.

Conclusion

The SARS-CoV-2 LC-MS Kit (RUO) offers a fully integrated, sensitive, and reproducible workflow for direct quantification of viral nucleocapsid peptides. Its automation, rapid runtime, and robust performance support harmonized research across laboratories, facilitating deeper insights into SARS-CoV-2 biology and host interactions.

Reference

• Fajnzylber J, Regan J, Coxen K, et al. SARS-CoV-2 Viral Load is Associated with Increased Disease Severity and Mortality. Nat Commun. 2020;11:5493.
• Surkova E, Nikolayevskyy V, Drobniewski F. False-positive COVID-19 Results: Hidden Problems and Costs. Lancet Respir Med. 2020;8(12):1167–1168.
• Cardozo KHM, Lebkuchen A, Okai GG, et al. Establishing a Mass Spectrometry-Based System for Rapid Detection of SARS-CoV-2. Nat Commun. 2020;11(1):6008.
• Van Puyvelde B, Van Uytfanghe K, Tytgat O, et al. Cov-MS: A Community-Based Template Assay for MS-Based Protein Detection in SARS-CoV-2 Patients. JACS Au. 2021;1(5):690–700.
• Renuse S, Vanderboom PM, Maus AD, et al. Development of MS-Based Targeted Assay for Direct Detection of SARS-CoV-2 from Clinical Specimens. medRxiv. 2020.2020.08.05.20168948.